High purity rna compositions and methods for preparation thereof

ABSTRACT

The invention relates to improved RNA compositions for use in therapeutic applications. The RNA compositions are particularly suited for use in human therapeutic application (e.g., in RNA therapeutics). The RNA compositions are made by inproved processes, in particular, improved in vitro-transcription (IVT) processes. The invention also relates to methods for producing and purifying RNA (e.g, therapeutic RNAs), as well as methods for using the RNA compositions and therapeutic applications thereof.

RELATED APPLICATIONS

This application is a continuation of international patent applicationserial number PCT/US2017/051674, filed Sep. 14, 2017, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. provisional application62/394,711, filed Sep. 14, 2016, the entire contents of each of whichare incorporated by reference herein in its entirety.

BACKGROUND OF INVENTION

The ability to design, synthesize and deliver a nucleic acid, e.g., aribonucleic acid (RNA) for example, a messenger RNA (mRNA) inside acell, has provided advancements in the fields of therapeutics,diagnostics, reagents and for biological assays. Many advancements arebeing made in the process of intracellular translation of the nucleicacid and production of at least one encoded peptide or polypeptide ofinterest.

mRNA has immense therapeutic potential in that mRNA therapeutics cantransiently express essentially any desired protein while avoiding theadverse effects of viral and DNA-based nucleic acid delivery approaches.Mammalian cells, in particular, human cells, however, contain sensors ofnucleic acids including RNA as part of the innate immune system—and itis desirable to avoid such sensing and immune response when developingmRNA therapeutics.

In theory, mRNAs produced via chemical synthesis hold promise as mRNAtherapeutics, however, the majority of the research in this importanttherapeutic area to date has focused on in vitro-transcribed (IVT) mRNA,as this enzymatic process facilitates the production of long RNAs, onthe order of 1-2 or more kB, the standard length of most mRNA molecules.

Early work showed that incorporation of modified nucleosides, inparticular, pseudouridine, reduced innate immune activation andincreased translation of mRNA, but residual induction of type Iinterferons (IFNs) and proinflammatory cytokines remained (Kariko et al.(2005) Immunity 23(2): 165-75). Progress was made towards theidentification of the contaminants in nucleoside-modified IVT RNAidentifying double-stranded RNA (dsRNA) as being at lease partiallyresponsible for innate immune activation. Removal of such contaminantsby high performance liquid chromatography (HPLC) resulted in reduced IFNand inflammatory cytokine levels and in turn, higher expression levelsin primary cells (Kariko et al. (2011) Nuc. Acids Res. 39:e142).Notably, unmodified mRNAs still induced high levels of cytokinesecretion although they were better-translated following HPLCpurification.

WO 2013/102203 describes an RNAse III treatment method used to removedsRNA from IVT mRNA for repeated or continuous transfection into humanor animal cells, in particular, for reprogramming of cells from onedifferentiation state to another. The method purports to result inpreparations having decreased levels of dsRNA and increased levels ofintact ssRNA, as evidenced by higher levels of reprogramming factors andless toxicity to cells. Such methods, however, are not compatible foruse in the preparation of mRNAs for therapeutic use, in particular, forhuman therapeutic use. RNAse III is known to digest ssRNA as well asdsRNA and in trying to remove dsRNA contaminants, the integrity of thedesired ssRNA product is necessarily jeopardized. Thus, there exists aneed for better understanding of the nature of contaminants inIVT-generated mRNA preparations, in order to better control for levelsand nature of contaminants in IVT preparations. There further exists aneed for improved methods of preparing mRNA for therapeutic use and forhigh purity compositions produced according to such methods.

SUMMARY OF INVENTION

The invention involves, at least in part, the discovery of novel methodsfor in vitro RNA synthesis and related products. The RNA transcriptsproduced by the methods described herein have enhanced properties whichresult in qualitatively and quantitatively superior compositionscomprising said RNA transcripts. The RNA transcripts produced by themethods described herein have enhanced properties particularly importantfor mRNA, IncRNA, and other therapeutic and diagnostic RNA uses, such asimproved immune silencing and better safety profiles.

In particular, IVT RNA compositions of the invention are substantiallyfree of certain undesirable contaminants routinely associated with theIVT process. Notably, however, the methods of the invention arrive atmRNA compositions suitable for therapeutic use by controlling the natureand levels of contaminants produced in the IVT reaction, i.e., thecontaminants are not made in the initial reaction, as contrasted toart-described methods which attempt to remove contaminants once theyhave been produced. Without being bound in theory, it is believed thatpreventing the production of unwanted contaminants in the IVT reactionfrom the outset provides for improved compositions having higher purityand potency, measurable, for example, in terms of increased translationfrom full-length, intact mRNA in the composition.

A composition comprising an in vitro-transcribed (IVT) RNA and apharmaceutically acceptable excipient, wherein the composition issubstantially free of reverse complement transcription product isprovided in some aspects of the invention. In some embodiments, lessthan about 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%,0.6%, or 0.55% of the total mass of the RNA in the composition isreverse complement transcription product. In some embodiments, less thanabout 1.0% of the mass of the RNA in the composition is reversecomplement transcription product. In some embodiments, less than about0.5% of the mass of the RNA in the composition is reverse complementtranscription product. In some embodiments, less than about 0.25% of themass of the RNA in the composition is reverse complement transcriptionproduct. In some embodiments, less than about 0.1% of the mass of theRNA in the composition is reverse complement transcription product. Insome embodiments, less than about 0.05% of the mass of the RNA in thecomposition is reverse complement transcription product. In someembodiments, less than about 0.01% of the mass of the RNA in thecomposition is reverse complement transcription product. In someembodiments, less than about 0.005% of the mass of the RNA in thecomposition is reverse complement transcription product. In someembodiments, less than about 0.001% of the mass of the RNA in thecomposition is reverse complement transcription product. In exemplaryembodiments, the mass of the RNA in the composition is determined by LC,J2 Elisa, RNase III, Gel electrophoresis with radiolabeled NTPs, LCMS+/−nuclease or chemical digestion, NMR using labelled NTPs,chemically/isotopically/radioactively etc. labelled NTPs, cells,biochemical means, RIG-I ATPase activity or MS or gel electrophoresis orother methods known in the art to be suitable for detection and/orquantitation of RNA in RNA-containing compositions.

In some embodiments the reverse complement transcription product isfully complementary with a region of the RNA which is the desired orintended IVT transcription produce (e.g., an mRNA, IncRNA, or other RNAgreater than 50 nucleotides in length intended for therapeutic use). Aproduct that is fully complementary with the RNA transcript isconsidered to have 100% complementarity (e.g., over the length of thereverse complement transcription product. In other embodiments thereverse complement transcription product is partially complementary witha region of the RNA transcript. In some embodiments the reversecomplement transcription product is 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary with a region of theRNA transcript. In yet other embodiments the reverse complement productis 70%-90%, 75%-90%, 80%-90%, 85%-90%, 90%-95%, 91%-95%, 92%-95%,930%-95%, 94%-95%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% complementarywith a region of the RNA transcript.

The skilled artisan will appreciate that unintended or undesireablereverse complement transcription products generated in an IVT reactioncan have complementarity not only to the RNA transcript which is theintended or desired product of the IVT reaction (e.g., an mRNA, IncRNA,or other RNA greater than 50 nucleotides in length intended fortherapeutic use) but also can have complementarity to a strand of theDNA template from which the intended or desired RNA transcript isproduced. Without being bound in theory, it is believed that certainunintended or undesireable transcription products contaminating IVT RNAcompositions that are reduced or eliminated according to the novelprocesses of the instant invention are transcribed from the intended ordesired RNA transcription product, there exists the possibility thatcertain unintended or undesireable transcription products contaminatingIVT RNA compositions may be transcribed from the DNA template used inthe IVT reactions. The latter supposition, while possible, does notappear to be evidenced by the data presented herein which demonstratesreverse complement transcription products predominantly complementary tothe 5′ UTR and/or polyA tail of mRNA transcripts, whereas reversecomplement transcription products complementary to portions (e.g.,sequence elements) of a DNA template not present in transcribed mRNA aresignificantly decreased and in some instances not detectable.

In other aspects the invention is a composition comprising an invitro-transcribed (IVT) RNA encoding a polypeptide of interest and apharmaceutically acceptable excipient, wherein the composition issubstantially free of cytokine-inducing RNA contaminant. In someembodiments, less than about 0.5% of the mass of the RNA in thecomposition is cytokine-inducing RNA contaminant. In some embodiments,less than about 0.25% of the mass of the RNA in the composition iscytokine-inducing RNA contaminant. In some embodiments, less than about0.1% of the mass of the RNA in the composition is cytokine-inducing RNAcontaminant. In some embodiments, less than about 0.05% of the mass ofthe RNA in the composition is cytokine-inducing RNA contaminant. In someembodiments, less than about 0.01% of the mass of the RNA in thecomposition is cytokine-inducing RNA contaminant. In some embodiments,less than about 0.005% of the mass of the RNA in the composition iscytokine-inducing RNA contaminant. In some embodiments, less than about0.001% of the mass of the RNA in the composition is cytokine-inducingRNA contaminant. In some embodiments, the mass of the RNA in thecomposition is determined by LC or MS or gel electrophoresis or othermethod known in the art.

In some embodiments the invention features a composition comprising anin vitro-transcribed (IVT) RNA encoding a polypeptide of interest and apharmaceutically acceptable excipient, wherein the composition hasreduced levels of cytokine-inducing RNA contaminant and/or reversecomplement transcription product.

In some embodiments of the compositions of the invention describedherein, the the mass of the sum total of cytokine-inducing RNAcontaminant and/or the reverse complement transcription product is lessthan about 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%,1%, 0.90%, 0.8%, 0.7%, 0.6%, or 0.55%, 0.5%, 0.45%, 0.4%, 0.35%, 0.3%,0.0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% of the totalmass of the RNA in the composition. In some embodiments the ratio of thecytokine-inducing RNA contaminant to RNA transcription product (e.g.,intended or desired RNA transcript) in the composition is 99:1, 95:5,90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55,40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, or 1:99. In otherembodiments the ratio of the reverse complement transcription product tothe RNA transcription product (e.g., intended or desired RNA transcript)in the composition is 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30,65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80,15:85, 10:90, 5:95, or 1:99.

The size of the contaminant may vary. In some embodiments the length ofthe cytokine-inducing RNA contaminant and/or RNA transcription productis greater than 2 nucleotides up to and including the length of the fulllength transcription product (e.g., the intended or desiredtranscription produce, for example the mRNA transcript). In otherembodiments the length of the cytokine-inducing RNA contaminant and/orRNA transcription product is greater than 5, 10, 15, 20, 25, 30, 35, 40,45 or 50 nucleotides each up to and including the length of the fulllength transcription product. In other embodiments the length of thecytokine-inducing RNA contaminant and/or RNA transcription product is2-500 nucleotides in length, 10-500 nucleotides in length, 15-500nucleotides in length, 20-500 nucleotides in length, 30-500 nucleotidesin length, 40-500 nucleotides in length, 50-500 nucleotides in length,100-500 nucleotides in length, 200-500 nucleotides in length, 300-500nucleotides in length, 400-500 nucleotides in length, 2-200 nucleotidesin length, 10-200 nucleotides in length, 15-200 nucleotides in length,20-200 nucleotides in length, 30-200 nucleotides in length, 40-200nucleotides in length, 50-200 nucleotides in length, 100-200 nucleotidesin length, 200-300 nucleotides in length, 300-400 nucleotides in length,2-100 nucleotides in length, 10-100 nucleotides in length, 15-100nucleotides in length, 20-100 nucleotides in length, 30-100 nucleotidesin length, 40-100 nucleotides in length, or 50-100 nucleotides inlength.

The skilled artisan will appreciate that RNA contaminants of a certainstructure and/or length are quite prone to stimulating undesired orunwanted immune responses, for example, RNA contaminants of at least 15or at least 20 or at least 25 nucleotides in length, in particular, RNAcontaminants that are double-stranded in nature (dsRNAs). Removal ofsuch contaminants is possible using certain art-recognized methodologies(e.g., enzymatic and/or purification processes or method steps).However, each of such additional purification process or step in thegeneration of, for example, mRNAs, IncRNA, or other RNA greater than 50nucleotides in length intended for therapeutic use, introduces thepossibility of reduced fidelity of the inended product, for example, bysubjecting the direct IVT reaction product to (1) enzymatic conditions(e.g., RNAse treatment producing fragments of RNA) and/or (2) hightemperature, non-physiologic solvent conditions (e.g., HPLC or RPchromatography conditions) which can compromise the quality of the RNAproduct in the process of attempting to degrade or remove contaminants.

In certain aspects of the invention, undesired or unwanted contaminantsare a population of RNA species having a distribution of, for example,sizes and masses within a certain range. For example, a certain type ofcontaminant can have less than 5% of any individual species ofcontaminant (i.e., RNA species of the same sequence, same length, etc.).In other embodiments contaminants can have have less than 4.5%, 4.0%,3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%,0.001%, 0.0005%, or 0.0001%, of any individual species.

In some embodiments the reverse complement transcription product is aRNA (dsRNA, ssRNA or a ds-ssRNA hybrid having a ds portion and a ssportion) comprising a strand comprising a sequence which is a reversecomplement of the IVT RNA or a RNA (dsRNA, ssRNA or a ds-ssRNA. In someembodiments, where the Poly A tail is encoded within a PolyA:T tractwithin the DNA template, the reverse complement product comprises astrand comprising a polyU sequence or others ways commonly used in theart to install polyA tails in RNA. The poly U sequence is, for example,pppU(U)n wherein n is 1 or greater. In some embodiments n is 1-100 (forencoded poly A tails where target RNA has 100 nt poly A tail. In otherembodiments n is greater than 30 or 30-200. In exemplary embodiments thereverse complement product initiates with a 5′ triphosphate (5′-PPP). Inother embodiments the reverse complement product initiates with a 5′diphosphate (5′-PP) or a 5′ monophosphate (5′-P).

In some embodiments the reverse complement transcription productcomprises a reverse complement of the 5′-end of the IVT RNA and/or areverse complement of the 3′-end of the IVT RNA. In some embodiments thereverse complement of the 5′-end of the IVT RNA comprises a sequencecomplementary to all or a portion of a 5′ UTR of the IVT RNA. In otherembodiments the reverse complement of the 3′-end of the IVT RNAcomprises a sequence complementary to all or a portion of a polyA tailof the IVT RNA. In yet other embodiments the reverse complement of the3′-end is the reverse complement of a tailless RNA. In yet otherembodiments the reverse complement transcription product comprises asequence complementary to all or a portion of a 5′ end, a 3′ end, anopen reading frame and/or a polyA tail of the RNA or any combinationthereof.

In exemplary aspects of the invention, a cytokine-inducingRNA-contaminant is a RNA (dsRNA, ssRNA or a ds-ssRNA. In someembodiments the cytokine-inducing RNA-contaminant is a strand that insome embodiments comprises a reverse sequence which is a reversecomplement of the IVT RNA or a dsRNA or ssRNA comprising a strandcomprising a polyU sequence.

In some embodiments the strand comprising the sequence which is thereverse complement of the IVT RNA or the strand comprising the polyUsequence initiates with a 5′ triphosphate (5′-PPP). In some embodimentsthe polyU sequence is greater than 20 nucleotides in length. In someembodiments the polyU sequence is greater than 30 nucleotides in length.In other embodiments the polyU sequence is single stranded. In yet otherembodiments the polyU sequence is double stranded.

In some embodiments the cytokine-inducing RNA-contaminant comprises areverse complement of the 5′-end of the IVT RNA and/or a reversecomplement of the 3′-end of the IVT RNA. In some embodiments the reversecomplement of the 5′-end of the IVT RNA comprises a sequencecomplementary to all or a portion of a 5′ UTR of the IVT RNA. In otherembodiments the reverse complement of the 3′-end of the IVT RNAcomprises a sequence complementary to all or a portion of a polyA tailof the IVT RNA. In some embodiments the reverse complement comprises asequence complementary the first 10-15 nucleotides of the 5′ UTR. Insome embodiments the reverse complement comprises a sequencecomplementary the first 10-20 nucleotides of the 5′ UTR. In someembodiments the reverse complement comprises a sequence complementarythe first 10-30 nucleotides of the 5′ UTR. In some embodiments thereverse complement comprises a sequence complementary the first 10-40nucleotides of the 5′ UTR. In yet other embodiments thecytokine-inducing RNA-contaminant comprises a sequence complementary toall or a portion of a 5′ end, a 3′ end, an open reading frame and/or apolyA tail of the RNA or any combination thereof.

In some embodiments the cytokine-inducing RNA-contaminant is a singlestranded tri-phosphate reverse complement of 20 nucleotides or greater.In other embodiments the cytokine-inducing RNA-contaminant is a singlestranded tri-phosphate reverse complement of 25 nucleotides or greater.In other embodiments the cytokine-inducing RNA-contaminant is a singlestranded tri-phosphate reverse complement of 30 nucleotides or greater.In some embodiments the single stranded tri-phosphate reverse complementis 20-200 nucleotides in length. In some embodiments the single strandedtri-phosphate reverse complement is 20-100 nucleotides in length. Insome embodiments the single stranded tri-phosphate reverse complement is20-50 nucleotides in length. In some embodiments the single strandedtri-phosphate reverse complement is 25-200 nucleotides in length. Insome embodiments the single stranded tri-phosphate reverse complement is25-100 nucleotides in length. In some embodiments the single strandedtri-phosphate reverse complement is 25-50 nucleotides in length. In someembodiments the single stranded tri-phosphate reverse complement is30-200 nucleotides in length. In some embodiments the single strandedtri-phosphate reverse complement is 30-100 nucleotides in length. Insome embodiments the single stranded tri-phosphate reverse complement is30-50 nucleotides in length.

In other embodiments the cytokine-inducing RNA-contaminant is a singlestranded reverse complement having a terminal tri-phosphate-A,tri-phosphate-C, or tri-phosphate-U.

In other embodiments the cytokine-inducing RNA-contaminant is a doublestranded tri-phosphate reverse complement of 20 nucleotides or greater.In some embodiments the double stranded tri-phosphate reverse complementhas 20-200 nucleotides. In yet other embodiments the cytokine-inducingRNA-contaminant is a double stranded tri-phosphate reverse complementthat is a perfect duplex (no single stranded regions). In otherembodiments the cytokine-inducing RNA-contaminant is a double strandedtri-phosphate reverse complement that includes a single strandedoverhang.

In some aspects of the invention, a dsRNA comprises strands of between20 and 100 nucleotides in length in some embodiments. In otherembodiments the dsRNA is of duplex of between about 20 and about 50 bpin length. In yet other embodiments the dsRNA comprises strands of1-1,000, 5-1,000, 10-1,000, 100-1,000, 500-1,000, 1-10, 1-20, 1-50,1-100, 5-10, 5-20, 5-30, 5-50, 5-100, 5-200, 5-300, 5-400, 10-20, 10-30,10-100, 10-200, 10-300, 10-400, 10-500, 20-25, 20-30, 20-100, 20-200,20-300, 20-400, 20-500, 30-35, 30-40, 30-100, 30-200, 30-300, 30-400, or30-500 nucleotides in length. In yet other embodiments the dsRNAcomprises strands of 1 nucleotide to full length transcript length.

In other embodiments less than about 0.5% of the mass of the RNA in thecomposition is dsRNA of a size greater than 40 base pairs.

The purity of the products may be assessed using known analyticalmethods and assays. In exemplary aspects of the invention the amount ofreverse complement transcription product or cytokine-inducing RNAcontaminant is determined by high-performance liquid chromatography(such as reverse-phase chromatography, size-exclusion chromatography),Bioanalyzer chip-based electrophoresis system, ELISA, flow cytometry,acrylamide gel, a reconstitution or surrogate type assay. The assays maybe performed with or without nuclease treatment (P1, RNase III, RNase Hetc.) of the RNA preparation. Electrophoretic/chromatographic/mass specanalysis of nuclease digestion products may also be performed.

In some embodiments the mass of RNA is determined by mass spectrometrysuch as LC-MS, MALDI-TOF (matrix-assisted laser desorption ionizationtime of flight).

In some embodiments the composition comprises contaminant transcriptsthat have a length less than a full length transcript, such as forinstance at least 100, 200, 300, 400, 500, 600, 700, 800, or 900nucleotides less than the full length. Contaminant transcripts caninclude reverse or forward transcription products (transcripts) thathave a length less than a full length transcript, such as for instanceat least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides lessthan the full length. Exemplary forward transcripts include, forinstance, abortive transcripts. In certain embodiments the compositioncomprises a tri-phosphate poly-U reverse complement of less than 30nucleotides. In some embodiments the composition comprises atri-phosphate poly-U reverse complement of any length hybridized to afull length transcript. In other embodiments the composition comprises asingle stranded tri-phosphate forward transcript. In other embodimentsthe composition comprises a single stranded RNA having a terminaltri-phosphate-G. In other embodiments the composition comprises singleor double stranded RNA of less than 12 nucleotides or base pairs(including forward or reverse complement transcripts). In any of theseembodiments the composition may include less than 50%, 45%, 400/%, 35%,30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% ofany one of or combination of these less than full length transcripts.

In other embodiments the RNA is produced by a process or is preparableby a process comprising

(a) forming a reaction mixture comprising a DNA template and NTPsincluding adenosine triphosphate (ATP), cytidine triphosphate (CTP),uridine triphosphate (UTP), guanosine triphosphate (GTP) and optionallyguanosine diphosphate (GDP), and (eg. buffer containing T7 co-factor eg.magnesium).

(b) incubating the reaction mixture under conditions such that the RNAis transcribed, wherein the concentration of at least one of GTP, CTP,ATP, and UTP is at least 2× greater than the concentration of any one ormore of ATP, CTP or UTP or the reaction further comprises a nucleotidediphosphate (NDP) or a nucleotide analog and wherein the concentrationof the NDP or nucleotide analog is at least 2× greater than theconcentration of any one or more of ATP, CTP or UTP, In some embodimentsthe ratio of concentration of GTP to the concentration of any one ATP,CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 orat least 6:1.

The ratio of concentration of GTP to concentration of ATP, CTP and UTPis, in some embodiments 2:1, 4:1 and 4:1, respectively. In otherembodiments the ratio of concentration of GTP to concentration of ATP,CTP and UTP is 3:1, 6:1 and 6:1, respectively. The reaction mixture maycomprise GTP and GDP and wherein the ratio of concentration of GTP plusGDP to the concentration of any one of ATP, CTP or UTP is at least 2:1,at least 3:1, at least 4:1, at least 5:1 or at least 6:1 In someembodiments the ratio of concentration of GTP plus GDP to concentrationof ATP, CTP and UTP is 3:1, 6:1 and 6:1, respectively.

In yet other embodiments the RNA is produced by a process or ispreparable by a process comprising

(a) forming a reaction mixture comprising a DNA template and adenosinetriphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate(UTP), guanosine triphosphate (GTP) and optionally guanosine diphosphate(GDP), and a buffer magnesium-containing buffer,

(b) incubating the reaction mixture under conditions such that the RNAis transcribed,

wherein the effective concentration of phosphate in the reaction is atleast 150 mM phosphate, at least 160 mM, at least 170 mM, at least 180mM, at least 190 mM, at least 200 mM, at least 210 mM or at least 220mM. The effective concentration of phosphate in the reaction may be 180mM. The effective concentration of phosphate in the reaction in someembodiments is 195 mM. In other embodiments the effective concentrationof phosphate in the reaction is 225 mM.

In other embodiments the RNA is produced by a process or is preparableby a process comprising

(a) forming a reaction mixture comprising a DNA template and adenosinetriphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate(UTP), guanosine triphosphate (GTP) and optionally guanosine diphosphate(GDP), and a buffer magnesium-containing buffer,

(b) incubating the reaction mixture under conditions such that the RNAis transcribed,

wherein the magnesium-containing buffer comprises Mg2+ and wherein themolar ratio of concentration of ATP plus CTP plus UTP pus GTP andoptionally GDP to concentration of Mg2+ is at least 1.0, at least 1.25,at least 1.5, at least 1.75, at least 1.85, at least 3 or higher. Themolar ratio of concentration of ATP plus CTP plus UTP pus GTP andoptionally GDP to concentration of Mg2+ may be 1.5. The molar ratio ofconcentration of ATP plus CTP plus UTP pus GTP and optionally GDP toconcentration of Mg2+ in some embodiments is 1.88. The molar ratio ofconcentration of ATP plus CTP plus UTP pus GTP and optionally GDP toconcentration of Mg2+ in some embodiments is 3.

In some embodiments the composition is produced by a process which doesnot comprise an dsRNase (e.g., RNaseIII) treatment step. In otherembodiments the composition is produced by a process which does notcomprise a reverse phase (RP) chromatography purification step. In yetother embodiments the composition is produced by a process which doesnot comprise a high-performance liquid chromatography (HPLC)purification step.

The RNA in some embodiments is modified mRNA. In other embodiments theRNA is unmodified RNA. In other embodiments the RNA is IncRNA. In yetother embodiments is RNA greater than 50 nucleotides in length. The RNAmay include a UTP and the UTP is modified UTP.

In some embodiments the amount of reverse complement transcriptionproduct or cytokine-inducing species is determined indirectly by aprocess comprising:

(a) producing a composition comprising a model RNA from a DNA templateencoding the model RNA under identical IVT conditions as used to producea IVT RNA, and

(b) determining the amount of reverse complement transcription productor cytokine-inducing species by LC-MS in the composition comprising themodel RNA,

wherein the amount of reverse complement transcription product orcytokine-inducing species by LC-MS in the composition comprising themodel RNA indicates the amount of reverse complement transcriptionproduct or cytokine-inducing species in the composition comprising theIVT RNA.

In other aspects the invention is an in vitro-transcribed (IVT) RNAcomposition wherein the RNA is not subject to RNaseIII treatments and/oris not subject to RP purification.

In yet other aspects the invention is a composition comprising an invitro-transcribed (IVT) single stranded RNA encoding a polypeptide ofinterest and a pharmaceutically acceptable excipient, wherein greaterthan 98% of the RNA is single stranded and wherein the single strandedRNA comprises transcripts of different lengths. In some embodiments thesingle stranded RNA comprising transcripts of different lengths includesfull length transcript and abortive transcripts. In some embodiments80-98% of the single stranded non-full length transcript comprisesabortive transcripts. In yet other embodiments 95-98% of the singlestranded non-full length transcript comprises abortive transcripts.

A unit of use composition is provided in other aspects of the invention.The unit of use composition is an in vitro-transcribed (IVT) singlestranded RNA encoding a polypeptide of interest and a pharmaceuticallyacceptable excipient, wherein the composition is free of residualorganic solvents.

In other aspects the invention is a composition comprising an invitro-transcribed (IVT) single stranded RNA encoding a polypeptide ofinterest and a pharmaceutically acceptable excipient, wherein thecomposition is non-immunogenic and wherein the single stranded RNAcomprises transcripts of different lengths. In some embodiments thesingle stranded RNA comprising transcripts of different lengths includesfull length transcript and fragment transcripts such as abortivetranscripts. Fragment transcripts include for instance non-full lengthsense RNAs, truncated or prematurely terminated transcripts as well asabortive transcripts which are typically less than the first 25nucleotides of a transcription product.

In other aspects, the invention is a method of preparing RNA comprising

(a) forming a reaction mixture comprising a DNA template and NTPsincluding adenosine triphosphate (ATP), cytidine triphosphate (CTP),uridine triphosphate (UTP), guanosine triphosphate (GTP) and optionallyguanosine diphosphate (GDP), and a buffer eg. a magnesium-containingbuffer, and

(b) incubating the reaction mixture under conditions such that the RNAis transcribed, wherein the concentration of at least one of GTP, CTP,ATP, and UTP is at least 2× greater than the concentration of any one ormore of ATP, CTP or UTP or the reaction further comprises a nucleotidediphosphate (NDP) or a nucleotide analog and wherein the concentrationof the NDP or nucleotide analog is at least 2× greater than theconcentration of any one or more of ATP, CTP or UTP. In some embodimentsthe ratio of concentration of GTP to the concentration of any one ATP,CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 orat least 6:1 to produce the RNA.

In some embodiments the ratio of concentration of GTP to concentrationof ATP, CTP and UTP is 2:1, 4:1 and 4:1, respectively. In otherembodiments the ratio of concentration of GTP to concentration of ATP,CTP and UTP is 3:1, 6:1 and 6:1, respectively. In yet other embodimentsthe reaction mixture comprises GTP and GDP and wherein the ratio ofconcentration of GTP plus GDP to the concentration of any one of ATP,CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 orat least 6:1 in other embodiments the ratio of concentration of GTP plusGDP to concentration of ATP, CTP and UTP is 3:1, 6:1 and 6:1,respectively.

Any of the compositions described herein may be a reaction mixture,e.g., a mixture of an IVT reaction that has not been purified by othermethods such as RP chromatography. In other aspects the compositions arefinal products ready for therapeutic administration to a subject.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a graph depicting the results of an IFN-β assay screening hEPOchemistry variants, nLuc and vehicle controls as well as short modelRNA-1 in BJ fibroblasts.

FIG. 2 shows the results of an LCMS analysis of a short modeltranscript. The model demonstrates that the abortive species are presentin all three chemistries. The top trace shows unmodified short modelRNA-1, the middle trace shows short model RNA with all uridines modifiedto pseudouridine and all cytidines modified with 5′O-methyl, and thebottom trace shows short model RNA1 with some uridine and cytidineresidues modified.

FIG. 3 shows the results of an LCMS analysis of a model transcript. Themodel demonstrates the impurity profiles of model RNA-4 and hEPOprepared by IVT.

FIG. 4 shows that T7 can be used to perform RNA-templated RNAtranscription in the absence of DNA template which upon treatmentconfers an immunostimulatory product.

FIGS. 5A and 5B show the impact of reverse-phase (RP) and IVT with anexcess of GTP on the amount of RNase III substrate. Both alpha processand RP purification reduce RIII substrate. An additive effect ofcombining both is shown. FIG. 5A shows a Capillary Electrophoresisanalysis of RNase III treated hEPO G5 material. FIG. 5B shows aCapillary Electrophoresis analysis of RNase III treated hEPO G0material.

FIG. 6 shows transfection data from hEPO protein expression and IFN-β.

FIG. 7 is a Capillary Electrophoresis analysis of a short transcripttranscribed using different processes and treated with RNase III. Thedata show the effect of model RNAs treated with RNase III.

FIGS. 8A and 8B show the results of the RP-IP purity method. FIG. 8Ashows model RNA-4 subjected to RNase III treatment following IVT usingthe equimolar method and FIG. 8B shows model RNA-4 subjected to RNaseIII treatment following IVT with an excess of GTP.

FIG. 9 is a RP Fractionation of hEPO treated with and without RNase III.

FIGS. 10A-10D show hEPO fraction RNase III fragment analyzer datafollowing equimolar (FIGS. 10A, 10B, and 10C) reactions. The hEPO wasmodified so that its uridine bases were 1-methylpseudouridine. Treatmentwith RNase III did not show appreciable purity differences using processwith excess GTP. With equimolar there is considerable substrate. FIG.10D shows in vitro IFNbeta analysis of hEPO EQ G5 untreated or afterRNase III treatment under equimolar conditions.

FIGS. 11A-11D show Capillary Electrophoresis analysis of RP fractionatedhEPO Alpha +/−RIII treatment. FIGS. 11A, 11B and 11C show the effects ofIVT with an excess of GTP in the reactions. FIG. 11D shows IVT withexcess GTP, which resulted in no IFN response.

FIG. 12 shows the results of a J2 anti-dsRNA ELISA assay.

FIG. 13 shows that dsRNA is removed by RNase III treatment.

FIG. 14 shows the results of an IVT characterization study, illustratingthat the IVT with an excess of GTP is less sensitive to lowtemperature-induced cytokine spikes.

FIG. 15 shows the nuclease P1 results of the IVT characterization study.

FIGS. 16A to 16B show an impurity analysis by LCMS of RNA-based IVT indifferent chemistries using G5 in Equimolar process (FIG. 16A) and alphaprocess (FIG. 16B).

FIG. 17 shows IFN-β in BJ fibroblasts under the different IVTconditions.

FIG. 18 shows that dsRNA cannot be capped by vaccinia.

FIG. 19 shows the effects of CIP treatment on different dsRNA species.

FIG. 20 shows FA purity data from the in vivo experiments.

FIG. 21 shows IFN-β induction in BJ fibroblasts.

FIG. 22 shows in vivo expression of hEPO.

FIGS. 23A to 23D show cytokine Luminex data from the in vivoexperiments.

FIG. 24 shows in vivo B-cell activation frequencies.

FIG. 25 shows the results of an IFN-β assay, analyzing short dsRNAs. Theassay is an in vitro analysis of short 5′ triphosphorylated oligos.

FIG. 26 shows the results of an IFN-β assay, analyzing 20mer and polyU/AdsRNAs.

FIG. 27 shows an analysis of 3′ overhang with respect to IFN-0 response.

FIG. 28 shows the results of a cytokine assay testing dsRNA standardswith 5′ overhang, perfect duplex, and 3′ overhang of varying lengths.

FIG. 29 is a graph depicting in vitro analysis of polyU species.

FIG. 30 is a graph depicting in vitro analysis of ssRNA oligo standards.

FIG. 31 is a graph depicting in vitro analysis of dsRNA oligos standardswith different 5′ functionalities.

FIG. 32 is a graph demonstrating that phosphatase cannot dephosphorylatedsRNA.

FIG. 33 is a graph demonstrating the ssRNA Impurity Dose Response(IFNbeta in BJ Fibroblasts).

FIG. 34 is a graph demonstrating the dsRNA Impurity Dose Response(IFNbeta in BJ Fibroblasts).

FIG. 35 is a graph demonstrating the IFNbeta Response for modified 5′nucleotide on Forward Oligo Standards.

FIG. 36 is a graph demonstrating the IFNbeta Response for modified 5′nucleotide on Reverse Complement Oligo Standards.

FIG. 37 is a graph demonstrating the IFNbeta Response for 5′ hydroxylfunctionalized dsRNA.

FIG. 38 is a graph demonstrating that that alpha process generates moreOH (clean) than equimolar process.

FIG. 39 is a graph showing calculated dsRNA for 1 ug mRNA.

FIG. 40 is a schematic depicting a traditional in vitro transcription(IVT) process and types of impurities formed.

DETAILED DESCRIPTION

In order to enhance methods for manufacturing protein-coding polymers,new methods of generating RNA have been developed. It has beendiscovered that changes can be made to an in vitro transcription processto produce an RNA preparation having vastly different properties fromRNA produced using a traditional in vitro transcription (IVT) process.The RNA preparations produced according to the methods of the invention(also referred to herein as the IVT RNA compositions) have propertiesthat enable the production of qualitatively and quantitatively superiorcompositions comprising said RNA transcripts. Even when coupled withextensive purification processes, RNA produced using traditional IVTmethods is qualitatively and quantitatively distinct from the RNApreparations of the invention. For instance, the RNA preparations of theinvention (and compositions comprising same) are less immunogenic incomparison to RNA preparations (and compositions comprising same) madeusing traditional IVT. The RNA preparations produced according to themethods of the invention (also referred to herein as the IVT RNAcompositions) further have properties that enable the production ofqualitatively and quantitatively superior protein production, forinstance, when translated. For instance, protein generated from the RNApreparations of the invention is less immunogenic in comparison to RNApreparations made using traditional IVT.

Additionally, increased protein expression levels with higher purity areproduced from the RNA preparations described herein. Although not boundby a mechanism, it is believed that substantial protein expressionlevels are a result of the high integrity of mRNA in the purifiedsamples. While some purification procedures can effectively remove alevel of contaminants by degradation of those contaminants, theintegrity of the pharmaceutical product is negatively impacted. Forinstance, it is asserted in prior art that RNAse digestion of mRNAsamples is useful for removing RNA contaminants. However, RNAsedigestion also reduces the integrity of the mRNA by degrading portionsof full length transcript produced by the IVT reaction. In contrast tothe prior art IVT/purification processes the integrity of mRNA using themethods of the invention is quite high because the methods produce verylittle to no double stranded transcripts that would require removalusing procedures such as RNAse digestion.

The RNA produced by the processes described herein is any RNA greaterthan 30 nucleotides in length which may be used for therapeutic ordiagnostic purposes. In some embodiments the RNA is an RNA of greaterthan 40, 50, 60, 75, 100, 200, 300, 400, 500, or 1,000 nucleotides inlength. In some embodiments the RNA is an RNA of greater than 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, or12,000 nucleotides in length. The RNA in some embodiments is mRNA. Insome embodiments the RNA is an RNA of about 500 to about 4000nucleotides in length, 1000 about to about 2000 nucleotides in length,750 about to about 1800 nucleotides in length, about 1500 to about 3000nucleotides in length, about 4000 to about 7000 nucleotides in length,or about 6000 to about 12000 nucleotides in length. The mRNA may bemodified or unmodified. In other embodiments the RNA is one or more ofthe following: mRNA, modified mRNA, unmodified RNA, IncRNA,self-replicating RNA, circular RNA, CRISPR guide RNA.

Traditional IVT reactions are performed by incubating a DNA templatewith an RNA polymerase and equimolar quantities of nucleotidetriphosphates, including GTP, ATP, CTP, and UTP in a transcriptionbuffer. An RNA transcript having a 5′ terminal guanosine triphosphate isproduced from this reaction. These reactions also result in theproduction of a number of impurities such as double stranded and singlestranded RNAs which are immunostimulatory and may have an additiveimpact. The methods of the invention which prevent formation of reversecomplements prevent the innate immune recognition of both species. Insome embodiments the methods of the invention result in the productionof RNA having significantly reduced T cell activity than an RNApreparation made using prior art methods with equimolar NTPs. The priorart attempts to remove these undesirable components using a series ofsubsequent purification steps. Such purification methods are undesirablebecause they involve additional time and resources and also result inthe incorporation of residual organic solvents in the final product,which is undesirable for a pharmaceutical product. It is labor andcapital intensive to scale up processes like reverse phasechromatography (RP): utilizing for instance explosion proof facilities,HPLC columns and purification systems rated for high pressure, hightemperature, flammable solvents etc. The scale and throughput for largescale manufacture are limited by these factors. Subsequent purificationis also required to remove alkylammonium ion pair utilized in RPprocess. In contrast the methods described herein even enhance currentlyutilized methods (eg RP). Lower impurity load leads to higherpurification recovery of full length RNA devoid of cytokine inducingcontaminants eg. higher quality of materials at the outset. Anadditional advantage of the modified IVT processes of the invention,when using RNase III as a preparative purification, is that since thereis less RNase III substrate, less inert/extraneous cleavage products(those that degrade but do not translate) are generated by RNase IIItreatment. If only trace amounts of dsRNA/RNase III substrate, eventhough may be cytokine silent, more final intact RNA product (intactcap/ORF/PolyA) capable of translating protein is present. This leads toa reduced burden for any subsequent purification.

It was discovered quite surprisingly, according to aspects of theinvention, that the manipulation of one or more of the reactionparameters in the IVT reaction produces a RNA preparation of highlyfunctional RNA without one or more of the undesirable contaminantsproduced using the prior art processes. One parameter in the IVTreaction that may be manipulated is the relative amount of a nucleotideor nucleotide analog in comparison to one or more other nucleotides ornucleotide analogs in the reaction mixture (e.g., disparate nucleotideamounts or concentration). For instance, the IVT reaction may include anexcess of a nucleotides, e.g., nucleotide monophosphate, nucleotidediphosphate or nucleotide triphosphate and/or an excess of nucleotideanalogs and/or nucleoside analogs. The methods of the invention producea high yield product which is significantly more pure than productsproduced by traditional IVT methods.

Nucleotide analogs are compounds that have the general structure of anucleotide or are structurally similar to a nucleotide or portionthereof. In particular, nucleotide analogs are nucleotides whichcontain, for example, an analogue of the nucleic acid portion, sugarportion and/or phosphate groups of the nucleotide. Nucleotides include,for instance, nucleotide monophosphates, nucleotide diphosphates, andnucleotide triphosphates. A nucleotide analog, as used herein isstructurally similar to a nucleotide or portion thereof but does nothave the typical nucleotide structure (nucleobase-ribose-phosphate).Nucleoside analogs are compounds that have the general structure of anucleoside or are structurally similar to a nucleoside or portionthereof. In particular, nucleoside analogs are nucleosides whichcontain, for example, an analogue of the nucleic acid and/or sugarportion of the nucleoside.

A nucleoside triphosphate, as used herein, refers to a moleculeincluding a nucleobase linked to a ribose (i.e. nucleoside) and threephosphates (i.e. nucleotide). A nucleotide diphosphate refers to thesame molecule, but which has two phosphate moieties. A nucleotidemonophosphate refers to the same molecule, but which has one phosphatemoiety. The nucleotide monophosphate, nucleotide diphosphate andtriphosphate are sometimes referred to herein as NMP, NDP and NTP,respectively. The N in NMP, NDP and NTP refer to any nucleotide,including naturally occurring nucleotides, synthetic nucleotides, andmodified nucleotides. Thus the terms NDP and NTP refer to nucleotidediphosphates and nucleotide triphosphates, respectively, having anynaturally occurring, synthetic, or modified nucleotide therein.

Natural nucleotide diphosphates include at least adenosine diphosphate(ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), anduridine diphosphate (UDP). Natural nucleotide triphosphates include atleast adenosine triphosphate (ATP), guanosine triphosphate (GTP),cytidine triphosphate (CTP), 5-methyluridine triphosphate (m5UTP), anduridine triphosphate (UTP). In some embodiments the NDP and/or NTP aremodified. For instance, modified NDP or NTP may have a handle to enableeasy purification and isolation.

Nucleotide triphophates are added to the RNA strand by a polymerase suchas T7 polymerase. Nucleotide diphosphates and monophosphates, incontrast can initiate the reaction (e.g., serve as the first transcribedmonomer) but won't be incorporated within the strand by T7 polymerase(e.g., won't be incorporated anywhere else in the strand). In someinstances the nucleotide diphophates, such as GDP, may be incorporatedas the first monomer. For instance if T7 initiates with GDP and producesa 5′GDP a functional RNA may be generated. 5′ GDP initiated RNA is stilla substrate for vaccinia capping enzyme. When an excess of NMP such asGMP is used in the reaction the purity may be enhanced by ligating a capon, as the transcriped product with 5′ PO4 is a substrate for ligase(s)(e.g., DNA/RNA ligase(s)).

The nucleotide analogs useful in the invention are structurally similarto nucleotides or portions thereof but, for example, are notpolymerizable by T7. Nucleotide/nucleoside analogs as used herein(including C, T, A, U, G, dC, dT, dA, dU, or dG analogs) include forinstance, antiviral nucleotide analogs, phosphate analogs (soluble orimmobilized, hydrolyzable or non-hydrolyzable), dinucleotide,trinucleotide, tetranucleotide, e.g., a cap analog, or aprecursor/substrate for enzymatic capping (vaccinia, or ligase), anucleotide labelled with a functional group to facilitateligation/conjugation of cap or 5′ moiety (IRES), a nucleotide labelledwith a 5′ PO4 to facilitate ligation of cap or 5′ moiety, or anucleotide labelled with a functional group/protecting group that can bechemically or enzymatically cleavable. Antiviral nucleotide/nucleosideanalogs include but are not limited to Ganciclovir, Entecavir,Telbivudine, Vidarabine and Cidofovir.

IVT Reaction Conditions

In exemplary aspects, the methods of the invention involve theproduction of RNA via an IVT reaction. IVT is an art-recognized methodused to generate synthetic polynucleotides in vitro. In vitrotranscribed (IVT) RNA can be engineered to transiently express proteinsby structurally resembling natural RNA. However, there are inherentchallenges of this drug class, particularly related to controlling thetranslational efficacy and immunogenicity of the IVT RNA. In particular,IVT RNA produces unwanted innate immune effects and there are verystringent purification procedures by HPLC that are typically applied asan additional, final RNA production step. The removal of minor amountsof short double stranded RNA fragments is critically important toachieve this further reduced immune response.

The typical reaction used in the prior art provides a high fidelityreasonably high yield product. However, the product has a baseline levelof contaminants, only some of which can be removed using routinepurification methods. The IVT reaction typically includes the following:an RNA polymerase, e.g., a T7 RNA polymerase at a final concentrationof, e.g., 1000-12000 U/mL, e.g., 7000 U/mL; the DNA template at a finalconcentration of, e.g., 10-70 nM, e.g., 40 nM; nucleotides (NTPs) at afinal concentration of e.g., 0.5-10 mM, e.g., 7.5 mM each; magnesium ata final concentration of, e.g., 12-60 mM, e.g., magnesium acetate at 40mM; a buffer such as, e.g., HEPES or Tris at a pH of, e.g., 7-8.5, e.g.40 mM Tris HCl, pH 8. In some embodiments 5 mM dithiothreitol (DTT)and/or 1 mM spermidine may be included. In some embodiments, an RNaseinhibitor is included in the IVT reaction to ensure no RNase induceddegradation during the transcription reaction. For example, murine RNaseinhibitor can be utilized at a final concentration of 1000 U/mL. In someembodiments a pyrophosphatase is included in the IVT reaction to cleavethe inorganic pyrophosphate generated following each nucleotideincorporation into two units of inorganic phosphate. This ensures thatmagnesium remains in solution and does not precipitate as magnesiumpyrophosphate. For example, an E. coli inorganic pyrophosphatase can beutilized at a final concentration of 1 U/mL.

A typical in vitro transcription reaction includes the following:

1 Template cDNA 1.0 μg 2 10x transcription buffer 2.0 μl (400 mMTris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT or TCEP, 10 mM Spermidine) 3Custom NTPs (25 mM each) 7.2 μl 4 RNase Inhibitor 20 U 5 T7 RNApolymerase 3000 U 6 dH₂0 Up to 20.0 μl and 7 Incubation at 37° C. for 1hr-5 hrs.

The crude IVT mix may be stored at 4° C. for 4-12 hours. One unit ofRNase-free DNase is then used to digest the original template. After 15minutes of incubation at 37° C., the RNA is purified using purificationtechniques such as dT resin, reverse phase HPLC or Ambion's MEGACLEAR™Kit (Austin, Tex.) following the manufacturer's instructions. Theexemplary IVT reaction is not limiting in terms of components or amountsof components used.

Similar to traditional methods, the RNA of the invention may also beproduced by forming a reaction mixture comprising a DNA template, andone or more NTPs such as ATP, CTP, UTP, GTP (or corresponding analog ofaforementioned components) and a buffer. The reaction is then incubatedunder conditions such that the RNA is transcribed. However, the methodsof the invention involve the surprising finding that the presence of anexcess amount of one or more nucleotides and/or nucleotide analogs canhave significant impact on the end product. The methods of the inventioncan be used to produce high quality product lacking unintended orundesireable impurities and without impacting the efficacy of thereaction.

The IVT methods of the invention involve a modification in the amount(e.g., molar amount or quantity) of nucleotides and/or nucleotideanalogs in the reaction mixture. In some aspects, one or morenucleotides and/or one or more nucleotide analogs may be added in excessto the reaction mixture. An excess of nucleotides and/or nucleotideanalogs is any amount greater than the amount of one or more of theother nucleotides such as NTPs in the reaction mixture. For instance, anexcess of a nucleotide and/or nucleotide analog may be a greater amountthan the amount of each or at least one of the other individual NTPs inthe reaction mixture or may refer to an amount greater than equimolaramounts of the other NTPs.

In the embodiment when the nucleotide and/or nucleotide analog that isincluded in the reaction mixture is an NTP, the NTP may be present in ahigher concentration than all three of the other NTPs included in thereaction mixture. The other three NTPs may be in an equimolarconcentration to one another. Alternatively one or more of the threeother NTPs may be in a different concentration than one or more of theother NTPs.

In some embodiments, the excess of the selected NTP is 2 times or fold(×), 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 15×-100×,10×-90×, 10×-80×, 10×-70× or even greater than the amount of any one ormore of the other individual NTPs in the reaction mixture. In otherembodiments, the excess of the selected NTP is 2 times or fold (×), 3×,4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 15×-100×, 10×-90×,10×-80×, 10×-70× or even greater than the amount of the total of theother individual NTPs in the reaction mixture. In exemplary embodiments,the NTP is in a molar excess relative to other NTPs in the reactionmixture. For example, the NTP in excess can be added at a molar ratio,e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, or greater than one or more of the other NTPs in thereaction mixture. In other embodiments, the excess of the selected NTPis in a concentration of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM,3.54.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100 mM, 120 mM, 150 mM, oreven greater than the amount of any one or more of the other individualNTPs in the reaction mixture or in a range of 60-100 mM or 4.5-100 mM.In other embodiments, the excess of the selected NTP is in aconcentration of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.54.0mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100 mM, 120 mM, 150 mM or evengreater than the amount of any one or more of the sum of the other NTPsin the reaction mixture.

In some instances, the NTP in excess in the reaction mixture is NTP-1and the other NTPs in the reaction mixture are NTP-2, NTP-3, and NTP-4.In some embodiments NTP-1 is present in the reaction mixture in agreater concentration than NTP-2, NTP-3, and NTP-4 and wherein NTP-2,NTP-3, and NTP-4 are each in an equimolar amount. In some embodimentsthe ratio of NTP-1:NTP-2:NTP-3:NTP-4 is at least 2:1:1:1, at least3:1:1:1, at least 4:1:1:1, at least 5:1:1:1, at least 6:1:1:1, at least7:1:1:1, at least 8:1:1:1, at least 9:1:1:1, at least 10:1:1:1, at least11:1:1:1, at least 12:1:1:1, at least 13:1:1:1, at least 14:1:1:1, atleast 15:1:1:1, at least 16:1:1:1, at least 17:1:1:1, at least 18:1:1:1,at least 19:1:1:1, each with a potential upper cap of NTP-1 as 20.

In some embodiments the ratio of NTP-1:NTP-2+NTP-3+NTP-4 is at least3:3, at least 5:3, at least 6:3, at least 7:3, at least 8:3, at least9:3, at least 10:3, or at least 15:3, each with a potential upper cap of20:3.

In other embodiments NTP-1 is present in the reaction mixture in agreater concentration than NTP-2, NTP-3, and NTP-4 and NTP-2 and NTP-3are each in an equimolar amount and NTP-4 is present in the reactionmixture in a concentration higher than NTP-2 and NTP-3 and less thanNTP-1. For instance, in some embodiments the ratio ofNTP-1:NTP-4:NTP-2:NTP-3 is at least 3:2:1:1, at least 4:3:1:1, at least4:2:1:1, at least 5:3:1:1, at least 5:3:2:2, at least 6:4:2:2, at least8:4:2:2, at least 9:2:1:1, at least 10:2:1:1, at least 11:2:1:1, atleast 12:2:1:1, at least 13:2:1:1, at least 14:2:1:1, at least 15:2:1:1,at least 16:2:1:1, at least 17:2:1:1, at least 18:2:1:1, at least19:2:1:1, each with a potential upper cap of NTP-1 as 20.

In other embodiments NTP-1 is present in the reaction mixture in agreater concentration than NTP-2, NTP-3, and NTP-4 and NTP-2 and NTP-3are each in an equimolar amount and NTP-4 is present in the reactionmixture in a concentration less than NTP-1, NTP-2 and NTP-3. Forinstance, in some embodiments the ratio of NTP-1:NTP-3:NTP-2:NTP-4 is atleast 3:2:2:1, at least 4:3:3:1, at least 4:2:2:1, at least 5:3:3:1, atleast 5:3:3:2, at least 6:4:4:2, at least 8:4:4:2, at least 9:2:2:1, atleast 10:2:2:1, at least 11:2:2:1, at least 12:2:2:1, at least 13:2:2:1,at least 14:2:2:1, at least 15:2:2:1, at least 16:2:2:1, at least17:2:2:1, at least 18:2:2:1, at least 19:2:2:1, each with a potentialupper cap of NTP-1 as 20.

NTP-1 in some embodiments is GTP, ATP, UTP, or CTP. NTP-2 in someembodiments is GTP, ATP, UTP, or CTP. NTP-3 in some embodiments is GTP,ATP, UTP, or CTP. NTP-4 in some embodiments is GTP, ATP, UTP, or CTP.

In some embodiments, the NTP is GTP and it is present in the mixture ata ratio of at least 2:1, at least 3:1, at least 4:1, at least 5:1, atleast 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, atleast 11:1, at least 12:1, at least 13:1, at least 14:1, or at least15:1 relative to the concentration of any one of ATP, CTP, or UTP. Theratio of GTP to other NTPs may be from about 2:1 to about 3:1, fromabout 2.5:1 to about 3.5:1, from about 3:1 to about 4:1, from about3.5:1 to about 4.5:1, from about 4:1 to about 5:1, from about 4.5:1 toabout 5.5:1, from about 5:1 to about 6:1, from about 5.5:1 to about6.5:1, from about 6:1 to 7:1, from about 6.5:1 to about 7.5:1, fromabout 7:1 to about 8:1, from about 7.5:1 to about 8.5:1, from about 8:1to about 9:1, from about 8.5:1 to about 9.5:1, and from about 9:1 toabout 10:1. In an embodiment, the ratio of concentration of GTP to theconcentration of ATP, CTP, and UTP may be 2:1, 4:1, and 4:1,respectively. In another embodiment, the ratio of concentration of GTPto the concentration of ATP, CTP, and UTP may be 3:1, 6:1, and 6:1,respectively.

In the embodiment when the nucleotide and/or nucleotide analog that isincluded in the reaction mixture is an NDP or a nucleotide analog, theNDP or nucleotide analog may be present in a higher concentration thanall four of the NTPs included in the reaction mixture. The four NTPs maybe in an equimolar concentration to one another. Alternatively one ormore of the four NTPs may be in a different concentration than one ormore of the other NTPs.

In other embodiments, the excess of the selected NDP or nucleotideanalog is 2 times or fold (×), 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×,12×, 13×, 14×, 15×, 15×-100×, 10×-90×, 10×-80×, 10×-70× or even greaterthan the amount of any one or more of the individual NTPs in thereaction mixture. In other embodiments, the excess of the selected NDPor nucleotide analog is 2 times or fold (×), 3×, 4×, 5×, 6×, 7×, 8×, 9×,10×, 11×, 12×, 13×, 14×, 15×, 15×-100×, 10×-90×, 10×-80×, 10×-70× oreven greater than the amount of the total of the individual NTPs in thereaction mixture. In exemplary embodiments, the NDP or nucleotide analogis in a molar excess relative to other NTPs in the reaction mixture. Forexample, the NDP or nucleotide analog in excess can be added at a molarratio, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,13:1, 14:1, 15:1, or greater than one or more of the NTPs in thereaction mixture. In other embodiments, the excess of the selected NDPor nucleotide analog is in a concentration of 0.5 mM, 1.0 mM, 1.5 mM,2.0 mM, 2.5 mM, 3.0 mM, 3.54.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM,8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100mM, 120 mM, 150 mM, or even greater than the amount of any one or moreof the individual NTPs in the reaction mixture or in a range of 60-100mM or 4.5-100 mM. In other embodiments, the excess of the selected NDPor nucleotide analog is in a concentration of 0.5 mM, 1.0 mM, 1.5 mM,2.0 mM, 2.5 mM, 3.0 mM, 3.54.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM,8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100mM, 120 mM, 150 mM or even greater than the amount of any one or more ofthe sum of the NTPs in the reaction mixture.

In some instances, the NTPs in the reaction mixture are NTP-1, NTP-2,NTP-3, and NTP-4. In some embodiments NDP or nucleotide analog ispresent in the reaction mixture in a greater concentration than NTP-1,NTP-2, NTP-3, and NTP-4 and wherein NTP-1, NTP-2, NTP-3, and NTP-4 areeach in an equimolar amount. In some embodiments the ratio of NDP ornucleotide analog:NTP-1+NTP+2:NTP-3+NTP-4 is at least 4:4, at least 5:4,at least 6:4, at least 7:4, at least 8:4, at least 9:4, at least 10:4,or at least 15:4, each with a potential upper cap of 20:4.

In some embodiments the excess of NDP or nucleotide analog is combinedwith an equivalent or greater concentration of one of the four NTPs.

In other embodiments NDP or nucleotide analog is present in the reactionmixture in a greater concentration than NTP-1, NTP-2, NTP-3, and NTP-4and NTP-1, NTP-2 and NTP-3 are each in an equimolar amount and NTP-4 ispresent in the reaction mixture in a concentration less than or greaterthan NTP-1, NTP-2 and NTP-3. For instance, in some embodiments the ratioof NDP or nucleotide analog:NTP-1:NTP-3:NTP-2:NTP-4 is at least3:2:2:2:2:1, at least 4:3:3:3:1, at least 4:2:2:2:1, at least 5:3:3:3:1,at least 5:3:3:3:2, at least 6:4:4:4:2, at least 8:4:4:4:2, at least9:2:2:2:1, at least 10:2:2:2:2:1, at least 11:2:2:2:2:1, at least12:2:2:2:1, at least 13:2:2:2:1, at least 14:2:2:2:1, at least15:2:2:2:1, at least 16:2:2:2:2:1, at least 17:2:2:2:2:1, at least18:2:2:2:2:1, at least 19:2:2:2:1, each with a potential upper cap ofNDP or nucleotide analog as 20.

In other embodiments NDP or nucleotide analog is present in the reactionmixture in a greater concentration than NTP-1, NTP-2, NTP-3, and NTP-4and NTP-2 and NTP-3 are each in an equimolar amount and NTP-1 and/orNTP-4 are present in the reaction mixture in a concentration less thanor greater than NTP-2 and NTP-3. In other embodiments NDP or nucleotideanalog is present in the reaction mixture in a greater concentrationthan NTP-1, NTP-2, NTP-3, and NTP-4 and NTP-1, NTP-2, NTP-3, and NTP-4are each present in a different amount from one another.

In some embodiments the upper limit of the excess of nucleotide ornucleotide analog in the reaction mixture is governed by the solubilitylimit.

In some embodiments the NTPs are salt NTPs. For instance the NTPs may beammonium NTPs, tris NTPs, lithium NTPs, potassium NTPs, or sodium NTPs.

In one embodiment of the invention, the IVT method may involve theaddition of a combination of NTP and NDP to the reaction mixture. TheNTP and NDP in combination may be added in excess to the reactionmixture. An excess of the combination of NTP and NDP is any amountgreater than the amount of one or more of at least one of the other NTPsor all of the other NTPs in the reaction mixture. For instance, anexcess of NTP and NDP may be the combined amount that is greater amountthan the amount of at least one of the other NTPs in the reactionmixture.

Thus, in some embodiments the IVT reaction may include an equimolaramount of nucleotide triphosphate relative to at least one of the othernucleotide triphosphates or less than an excess of nucleotidetriphosphate when it is used in combination with a correspondingnucleotide diphosphate, as long as the total amount of that nucleotideis present in excess in the reaction. A corresponding nucleotidediphosphate refers to a nucleotide diphosphate having the same base asthe nucleotide triphosphate. For example, the nucleotide triphosphatemay be GTP and the nucleotide diphosphate may be GDP.

In some embodiments, the NTP and NDP in combination are equimolar. Inanother embodiment, the amount of NTP is greater than the amount of NDPin the combination added to the reaction mixture. The amount of NDP maybe greater than the amount of NTP in the combination added to thereaction mixture. In some embodiments, the excess of the NTP and NDPcombination mixture is 2 times or fold (×), 3×, 4×, 5×, 6×, 7×, 8×, 9×,10×, 11×, 12×, 13×, 14×, 15×, 15×-100×, 10×-90×, 10×-80×, 10×-70× oreven greater than the amount of the other individual NTPs in thereaction mixture. In each embodiment the other individual NTPs may bepresent in the same (equimolar) or different amounts in the reactionmixture. The fold difference described herein refers to a comparisonwith at least one, at least two or all three of the other NTPs in thereaction mixture.

In other embodiments, the NTP is 2 times or fold (×), 3×, 4×, 5×, 6×,7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 15×-100×, 10×-90×, 10×-80×,10×-70× or even greater than the amount of the NDP in the reactionmixture. In yet other embodiments, the NDP is 2 times or fold (×), 3×,4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 15×-100×, 10×-90×,10×-80×, 10×-70× or even greater than the amount of the NTP in thereaction mixture.

In each of the embodiments described herein, the NTP and NDP may be, forexample, GTP and GDP, respectively, and may be present in the mixture ina concentration at least 6 times or fold (×), 7×, 8×, 9×, 10×, 11×, 12×,13×, 14×, 15×, 15×-100×, 10×-90×, 10×-80×, 10×-70× or even greater thanthe amount of any one of ATP, CTP, or UTP in the reaction mixture. Inexemplary embodiments, the NTP and NDP in combination are in a molarexcess relative to the other individual NTPs in the reaction mixture.For example, the NTP and NDP combination mixture can be added at a molarratio, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,13:1, 14:1, 15:1, or greater in the reaction mixture. The ratio of GTPand GDP to other NTPs may be from about 2:1 to about 3:1, from about2.5:1 to about 3.5:1, from about 3:1 to about 4:1, from about 3.5:1 toabout 4.5:1, from about 4:1 to about 5:1, from about 4.5:1 to about5.5:1, and from about 5:1 to about 6:1. In one embodiment, the ratio ofconcentration of GTP to the concentration of ATP, CTP, and UTP may be3:1, 6:1, and 6:1, respectively.

In other embodiments the ratio of NTP to NDP and in some embodiments GTPto GDP is considered relative to the ratio or purine nucleotide topyrimidine nucleotide (Pu:Py) in the reaction mixture. In someembodiments the GTP:GDP to Pu:Py ratios are 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or greater in thereaction mixture.

In some embodiments, the buffer contains phosphate. The effectiveconcentration of the phosphate is at least 150 mM, at least 160 mM, atleast 170 mM, at least 180 mM, at least 190 mM, at least 200 mM, atleast 210 mM, at least 220 mM, or at least 230 mM phosphate. In oneembodiment, the effective concentration of phosphate is 180 mM. Inanother embodiment, the effective concentration of phosphate is 195 mM.

In another embodiment, the buffer contains magnesium. The buffer mayhave a ratio of the the concentration of ATP plus CTP plus UTP plus GTPand optionally, GDP to molar concentration of Mg²⁺ of at least 1.0, atleast 1.1, at least 1.2, at least 1.25, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.75, at least 1.8, andat least 1.85 or 3. In other embodiments the ratio is 1.0, 1.1, 1.2,1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.85, 2, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, or 3 or any range of these variables. In oneembodiment, the ratio is 1.5. In another embodiment, the ratio is 1.88.In one embodiment, the ratio is 3.

In exemplary aspects of the invention, the IVT reaction (reactionmixture) includes an RNA polymerase, for example, T7, SP6, T3, etc. Insome embodiments, the polymerase, e.g., T7 polymerase is included at aconcentration of greater than 5 U/μl, greater than 10 U/μl, greater than20 U/μl, greater than 50 U/μl, or greater than 100 U/μl. In someembodiments the polymerase, e.g., T7 polymerase, concentration is arange of from about 1 to about 250 U/μl of reaction mixture, e.g., fromabout 1 to about 100 U/l or from about 100 to about 250 U/μl. In someembodiments, the T7 polymerase concentration is a range of from about 30to about 60 U/μl, about 60 to about 80 U/μl, about 80 to about 100 U/μl,about 100 to about 150 U/μl or from about 150 to about 200 U/μl. In someembodiments, the polymerase, e.g., T7 polymerase is included at aconcentration of 7, 14, 25, 50, 75, or 140.

As used herein, a DNA template refers to a polynucleotide template forRNA polymerase. The DNA template useful according to the methodsdescribed herein includes in some embodiments a gene of interest codingfor, e.g., a polypeptide of interest. The DNA template in someembodiments includes a RNA polymerase promoter, e.g., a T7 promoterlocated 5′ to and operably linked to the gene of interest and optionallya sequence coding for a poly A tail located 3′ to the gene of interest.

RNA polymerases known in the art may be used in the methods of thepresent invention. RNA polymerases include but are not limited to, aphage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, anSP6 RNA polymerase, and/or mutant polymerases such as, but not limitedto, polymerases able to incorporate modified nucleic acids. As anon-limiting example, the RNA polymerase may be modified to exhibit anincreased ability to incorporate a 2′-modified nucleotide triphosphatecompared to an unmodified RNA polymerase.

As used herein, “gene of interest” refers to a polynucleotide whichencodes a polypeptide or protein of interest. Depending on the context,the gene of interest refers to a deoxyribonucleic acid, e.g., a gene ofinterest in a DNA template which can be transcribed to an RNAtranscript, or a ribonucleic acid, e.g., a gene of interest in an RNAtranscript which can be translated to produce the encoded polypeptide ofinterest in vitro, in vivo, in situ or ex vivo. A polypeptide ofinterest includes but is not limited to, biologics, antibodies,vaccines, therapeutic proteins or peptides, etc.

An “RNA transcript” refers to a ribonucleic acid produced by an IVTreaction using a DNA template and an RNA polymerase. In some embodimentsthe RNA transcript is an mRNA and typically includes the coding sequencefor a gene of interest and a poly A tail. RNA transcript includes anmRNA. The RNA transcript can include modifications, e.g., modifiednucleotides. As used herein, the term RNA transcript includes and isinterchangeable with mRNA, modified mRNA “mmRNA” or modified mRNA, andprimary construct.

Purity

RNA produced according to the methods of the invention is surprisinglypure and of high integrity. It has fewer contaminants than RNApreparations produced according to traditional IVT methods. In someembodiments it has fewer immune stimulating contaminants than RNApreparations produced according to traditional IVT methods. Thecontaminants are RNA fragments produced by the reaction other than thedesired RNA. In some embodiments the RNA fragment contaminants arereverse complement transcription products and/or cytokine inducing RNAcontaminants. In other embodiments the RNA fragment contaminants aredouble stranded RNA or immunogenic contaminants.

The RNA preparations of the invention in some embodiments have lesscontaminants than RNA preparations produced according to traditional IVTmethods. In some embodiments the RNA preparations of the invention haveat least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, or 99% lesscontaminants than RNA preparations produced according to traditional IVTmethods. In other embodiments the RNA preparations of the invention aresubstantially free of contaminants. In other embodiments the RNApreparations of the invention are 100% free of contaminants.

Thus, the invention in some aspects involves the preparation of an IVTRNA that is substantially free of reverse complement transcriptionproduct without the need for further purification steps. As used herein,the term “reverse complement transcription product” refers to an RNAmolecule resulting from RNA-templated transcription. The reversecomplement transcription product may be in some embodiments is anRNA-templated transcription product. Without being bound in theory it isbelieved that the reverse complement product is predominantly or allRNA-templated transcription product. If the reverse complement productwere composed of DNA-templated transcription product, the product wouldinclude, for example, nucleotide sequences complementary to the T7promoter region from the DNA template. The reverse complement productscharacterized to date are predominantly free of sequences complementaryto, for example, the T7 promoter region. In some embodiments the reversecomplement transcription product is a double-stranded RNA (dsRNA), whichmay include one strand encoding a sequence which is a reverse complementof at least a portion of the IVT RNA. In other embodiments the reversetranscription complement product may be a dsRNA with one strandcomprising a polyU sequence. Either the reverse complement strandencoding the polypeptide of interest or the strand encoding the polyUsequence may initiate with a 5′ triphosphate (5′ppp). The RNA-templatetranscription product may include a reverse complement of the 5′-end ofthe IVT RNA and/or a reverse complement of the 3′-end of the IVT RNA.Furthermore, the reverse complement of the 5′-end of the IVT RNA may becomplementary to all or a portion of a 5′UTR of the IVT RNA. The reversecomplement may comprise a sequence complementary to the first 10-15, thefirst 5-15, the first 5-20, the first 10-20, the first 15-25 nucleotidesof the 5′UTR. Likewise, the reverse complement of the 3′-end of the IVTRNA may be complementary to all or a portion of a polyA tail of the IVTRNA. The reverse complement transcription product can be templated fromanywhere on the RNA and thus can be any size or complementary to anylocation on the template. For instance, the reverse complement productmay be a 5-mer 10-mer 15-mer 20-mer 25-mer 40-mer 50-mer 60-mer 70-mer,100-mer, 200-mer, etc. all the way up to the full length of the intendedor desired product.

The present invention features compositions comprising an IVT RNA and apharmaceutically acceptable excipient substantially free of reversecomplement transcription product. In some embodiments, in the IVT RNAthat is substantially free of reverse complement transcription productincludes reverse complement transcription product that makes up lessthan about 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%,1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.55%, 0.5%, 0.45%, 0.4%, 0.35%, 0.3%,0.0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% of the massof the total RNA. The mass of the RNA composition may be determined byany means known in the art. Examples of methods for determining the massof the RNA include liquid chromatography and mass spectrometry.

Although not bound in theory, it is believed that in some embodiments ofthe invention the contaminants are single-stranded reverse complementsbound to a population of the IVT RNAs forming a double strandedstructure in the context of longer RNAs. T7 may template off of anabortive (sense strand) nascent RNA as well as full length productnascent RNA. The RNA-templated transcript (antisense), once transcribed(initiating predominantly with pppC, pppU, and pppA), presumably remainsassociated with the nascent sense RNA. Alternatively if such constructsdo form they may be intrinsically silent. The association of the 2strands effectively is dsRNA with 5′ppp. The presence of 5′ppp on one ormore of the hybridized strands, renders the structure immunostimulatory.Even when the antisense RNA templated transcript is dissociated from theRNA, the presence of ssRNA with pppC, pppU, and pppA is still cytokineinducing. Since the methods of the invention produce RNA devoid of dsRNAas seen in J2 ELISA and RNase III treatment, the products would notassume a structure of large full length RNA. It is likely that the RNAis folding similarly if transcribed with equimolar or the inventive IVTprocess.

In some embodiments the RNA preparations of the invention aresubstantially free of cytokine-inducing RNA contaminant. As used herein,the term “cytokine-inducing contaminant” refers to an RNA molecule whichinduces cytokine generation, for example, type I interferon (IFNα/βinduction), for example, as determined in a cell-based cytokineinduction assay, for example, as determined in a BJ fibroblast/IFNbetaassay and/or Luminex assay as described in the working examples of theinstant specification. In exemplary aspects of the invention, the term“cytokine-inducing” contaminant refers to an RNA molecule which inducescytokine induction and which is substantially double-stranded in nature.

Without being bound in theory, it is believed that double-stranded RNAmolecules which result from aberrant polymerase transcription, forexample, transcription templated off the desired RNA produced in an IVTreaction, induce cytokines via activation of an innate immune responseakin to the natural antiviral immune response and includes two types ofpathogen recognition receptors (PRRs): the Toll-like receptors (TLRs)and the RIG-I-like receptors (RLRs), for example, toll-like receptor 3(TLR3), as well as the RNA helicases, for example, RIG-I and MDA5.Examples of other cytokine-inducing molecules include RNaseIIIsubstrates. An RNase III substrate, as used herein, refers to a doublestranded RNA molecule which is susceptible to cleavage by an RNase IIIenzyme.

In some embodiments, the cytokine-inducing RNA contaminant may be adouble-stranded RNA with a reverse sequence complementary to the IVT RNAor a polyU sequence. The reverse complement of the IVT RNA or the polyUsequence may initiate with a 5′ppp.

The cytokine-inducing RNA contaminant may include a reverse complementof the 5′-end of the IVT RNA and/or a reverse complement of the 3′-endof the IVT RNA. Furthermore, the reverse complement of the 5′-end of theIVT RNA may be complementary to all or a portion of a 5′UTR of the IVTRNA. The reverse complement may comprise a sequence complementary to thefirst 10-15, the first 5-15, the first 5-20, the first 10-20, the first15-25 nucleotides of the 5′UTR. In some embodiments the reversecomplement may comprise a sequence complementary to a range of 1-20,1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-200, 1-300, 1-400,1-500, 1-600, 1-700, 1-800, 1-900, 1-1000, 1-2000, 1-2500, or 1-3000nucleotides in length within the 5′UTR. In other embodiments the reversecomplement may comprise a sequence complementary to a range of 10-20,10-30, 10-40, 10-50, 1-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300,10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-2000,10-2500, or 10-3000 nucleotides in length within the 5′UTR. In otherembodiments the reverse complement may comprise a sequence complementaryto a range of 20-25, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90,20-100, 20-200, 20-300, 20-400, 20-500, 20-600, 20-700, 20-800, 20-900,20-1000, 20-2000, 20-2500, or 20-3000 nucleotides in length within the5′UTR. Likewise, the reverse complement of the 3′-end of the IVT RNA maybe complementary to all or a portion of a polyA tail of the IVT RNA. Thereverse complement may comprise a sequence complementary to the first10-15, the first 5-15, the first 5-20, the first 10-20, the first 15-25nucleotides of the 3′UTR. In some embodiments the reverse complement maycomprise a sequence complementary to a range of 1-20, 1-30, 1-40, 1-50,1-60, 1-70, 1-80, 1-90, 1-100, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700,1-800, 1-900, 1-1000, 1-2000, 1-2500, 1-3000, or 1-full length ormaximum size of the RNA nucleotides in length within the 3′UTR. In otherembodiments the reverse complement may comprise a sequence complementaryto a range of 10-20, 10-30, 10-40, 10-50, 1-60, 10-70, 10-80, 10-90,10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900,10-1000, 10-2000, 10-2500, or 10-3000 nucleotides in length within the3′UTR. In other embodiments the reverse complement may comprise asequence complementary to a range of 20-25, 20-30, 20-40, 20-50, 20-60,20-70, 20-80, 20-90, 20-100, 20-200, 20-300, 20-400, 20-500, 20-600,20-700, 20-800, 20-900, 20-1000, 20-2000, 20-2500, or 20-3000nucleotides in length within the 3′UTR.

The present disclosure includes a composition comprising an IVT RNA anda pharmaceutically acceptable excipient substantially free ofcytokine-inducing RNA contaminant. In some embodiments, thecytokine-inducing RNA contaminant makes up less than 0.5%, 0.45%, 0.4%,0.35%, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%of the mass of the RNA. The mass of the RNA composition may bedetermined by any means known in the art. Examples include liquidchromatography and mass spectrometry.

The dsRNA of the contaminant such as the cytokine-inducing RNAcontaminant and/or the reverse complement transcription product may be20 to 50 nucleotides in length. In other embodiments, the dsRNA may be20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70,70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110, 110-120, 120-130,130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225,225-250, 250-275, 275-300, 300-325, 325-350, 350-375, 375-400, 400-425,425-450, 450-475, 475-500, 500-550, 550-600, 600-650, 650-700, 700-750,750-800, 800-850, 850-900, 900-950, and 950-1000 nucleotides in length.In some embodiments, the mass of the dsRNA is greater than 40 base pairsand makes up less than about 0.5% of the RNA composition.

The contaminant strands may have 5′ppp ends. In some embodiments, thecontaminant strands may have a lower abundance of pppA, pppC, and pppU,as compared to equimolar process-produced RNA. In another embodiment,the contaminant strands may have lower ratios of pppA:pppG, pppC:pppG,and/or pppU:pppG as compared to equimolar processes. pppNTPs may bedetected by LC-MS following total nuclease digestion e.g. Nuclease P1treatment. Nuclease P1 digests RNA and DNA into single nucleotides. Theonly triphosphate species that should be present are for the initiatingnucleotides. If no RNA templated transcription products are formed,5′PPPG is the only triphosphate that should be present as this is theonly targeted site of initiation. Presence and abundance of 5′pppA,5′pppC, and/or 5′pppU as detected by LC/MS following Nuclease P1digestion are indicative of RNA templated RNA transcripts.

In addition to having less impurities, particularly double strandedimpurities, the IVT RNA compositions have a high proportion of fulllength functional RNA transcript relative to other RNA species in thecomposition, particularly when compared to traditional purified RNAcompositions produced using IVT methods combined with purification stepssuch as reverse phase chromatography or RNAse III treatment. In someembodiments greater than about 80%, 85%, 900/%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5% or 99.8% of the mass of the RNA comprisessingle stranded full length transcripts. In addition to the singlestranded full length transcripts, the IVT RNA composition may includeother single stranded RNA species such as single stranded partial RNAtranscripts in a sense orientation, including abortive transcripts. TheIVT RNA composition, however, is substantially free of RNAse IIIinsensitive fragments.

The RNA compositions described herein may include other componentsbesides the full length RNA transcript, e.g., truncated transcriptsand/or run-on transcripts. For instance, the RNA may include transcriptsof different lengths, e.g., shorter or longer than the full-lengthtranscript. Thus, in some embodiments the RNA preparation of theinvention includes truncated and/or abortive transcripts. RNA polymerasebinds to a DNA promoter and synthesizes the short mRNA transcripts. Asused herein, the term “truncated transcripts” refers to transcriptshaving identity to the IVT RNA, but being of insufficient length andlacking all required elements to encode the polypeptide of interest (egpoly A). In certain instances, truncated transcripts are released priorto the transcription complex leaving the promoter, termed abortivetranscripts. As used herein, the term “abortive transcripts” refers totranscripts having identity to the IVT RNA, but being of insufficientlength and lacking all required elements to encode the polypeptide ofinterest (eg poly A), generally having a length of 15 nucleotides orless. In exemplary aspects of the invention, the truncated and/orabortive transcripts are present and are not cytokine-inducing. In anembodiment, the truncated and/or abortive transcripts are removed fromthe sample. In some embodiments truncated transcripts have a length of100 nucleotides or less.

The methods of the instant invention also have been determined toproduce compositions having reduced 3′ heterogeneity or 3′ endheterogeneity, also referred to herein as increased 3′ homogeneity, or3′ end homogeneity. It was determined by the present inventors thattraditional equimolar IVT reaction conditions can produce transcriptsterminating at different 3′ residues (e.g., transcription not uniformlyterminating). An assay featured in the Working Examples was developed todetect the 3′ end heterogeneity resulting from traditional IVT reactions(the assay differentiating between non-A nucleobases occurring at the 3′end of a particular test transcript). Notably, the methods of theinstant invention produce transcripts having a lower degree of 3′ endheterogeneity (or more homogeneous 3′ ends). For example, transcriptsproduced according to traditional IVT reactions (e.g., equimolarreactions) can produce compositions in which greater than 50% of thetranscripts (optionally greater than 60%, greater than 70%, greater than75%, greater that 80% or more) have different ends, whereas transcriptsproduced according to the IVT reactions of the invention (e.g., alphareactions) can produce compositions in which less than 50% of thetranscripts, i.e., greater than 50% of the transcripts have the sameends, i.e., terminate at the same nucleobase (e.g., relative to the DNAtemplate) (optionally less than 40%, less that 30%, less than 25%, lessthan 20% or less) have different ends).

The truncated transcripts within the population of single strandedpartial RNA transcripts may include a range of sizes. For instance, insome embodiments, at least 80% of the population of truncatedtranscripts have a length of 100 nucleotides or less. In otherembodiments at least 50%, 60%, 70%, 85%, 90%, 95%, 98% or 100% of thepopulation of truncated transcripts have a length of 100 nucleotides orless.

The single stranded RNA population within the IVT RNA compositionsdescribed herein typically is free or substantially free of RNAse IIIinsensitive fragments. An “RNAse III insensitive fragment” as usedherein refers to single stranded transcripts having identity to the IVTRNA (sense orientation), but being of insufficient length and lackingall required elements to encode the polypeptide of interest (having lessnucleotides than full length transcripts) and wherein the fragment isproduced by enzymatic, in particular RNAse III, cleavage. The productionof RNAse III insensitive fragments can result, for example, in atraditional IVT process (as depicted in FIG. 40) coupled with an RNAseIII digestion.

As shown in FIG. 40 a first step in a traditional IVT/RNAse IIIpurification process involves a transcription reaction utilizing lineardsDNA template, equimolar concentrations of NTPs and RNA polymerase inthe presence of Mg²′. The reaction produces a mixed population of singlestranded truncated/abortive transcripts, full length RNA transcript,run-on transcripts and reverse complement impurities. The reversecomplement impurities can bind to some of the single stranded RNA or toother impurities, e.g., truncated transcripts, producing double strandedRNA and/or RNA having both double and single stranded regions. It hasbeen postulated in the art that RNAse III can be used to degrade thedouble stranded RNA from IVT compositions, thus effectively removing itfrom the composition. However, RNAse III can also degrade doublestranded regions of full length RNA transcript and/or run-on transcripts(e.g., double stranded regions resulting from reverse complementsbinding within polyA tail regions), leaving single stranded fragmentshaving a length of less than the full length RNA transcripts. Thesesingle stranded fragments are the RNAse III insensitive fragmentsdescribed herein. As a result of this RNAse degradation significantamounts of full length transcripts generated during the IVT process arelost, causing significant loss of product integrity. These compositionshave significantly lower ability to express protein when delivered to acell or subject.

RNAse III insensitive fragments generated following RNAse III treatmentof products generated according to methods such as those depicted inFIG. 40 may include a range of sizes. For instance, in some embodiments,at least 80% of the population of abortive transcripts have a length ofgreater than 100 nucleotides. In other embodiments at least 50%, 60%,70%, 85%, 90%, 95%, 98% or 100% of the population of RNAse IIIinsensitive fragments have a length of greater than 100 nucleotides.

Without being bound in theory, it is believed that the removal ofcertain species or contaminants, for example, dsRNA species orcontaminants, is important in the preparation of IVT RNA compositionsfor therapeutic use. By contrast, the presence of residual truncatedand/or abortive transcripts in IVT RNA compositions is not believed tobe required; such species are not believed to induce unwanted cytokinesand/or an innate immune response to the IVT RNA. In other embodimentsthe RNA preparation of the invention is substantially free of truncatedor abortive transcripts.

Although truncated/abortive transcripts may be present in the IVT RNAcompositions or the invention, RNAse III insensitive fragments are notpresent in the IVT RNA compositions because the composition is nottreated with RNAse III. While truncated transcripts and RNAse IIIinsensitive fragments both have a range of sizes or lengths, the averagelength of truncated transcripts is less than the average length of RNAseIII insensitive fragments. As such, when the composition comprises apopulation of single stranded partial RNA transcripts in a senseorientation and greater than 80% of the population of single strandedpartial RNA transcripts in a sense orientation has a nucleotide lengthof 100 nucleotides or less. In some embodiments greater than 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 990% or 100% of the population ofsingle stranded partial RNA transcripts in a sense orientation has anucleotide length of 100 nucleotides or less. In other embodimentsgreater than 50%, 55%, 60%, 65%, 70%, 75%, 85%, or 88% of the populationof single stranded partial RNA transcripts in a sense orientation has anucleotide length of 100 nucleotides or less.

In some embodiments the RNA preparation is a pharmaceutical compositionwith a pharmaceutically acceptable carrier. In other embodiments the RNApreparation is a reaction product (e.g., IVT reaction product) which hasnot yet been subjected to further purification techniques. The RNApreparation may include a number of other components in addition to theRNA. The reaction product, however, is substantially free of reversecomplement transcription product and/or cytokine inducing RNAcontaminants.

Assays

The amount of contaminant, including reverse complement transcriptionproduct and/or cytokine-inducing RNA contaminant, may be determined bymethods known in the art. Many methods for determining the purity of anucleic acid sample are known in the art. Exemplary methods include, butare not limited to, the following: high-performance liquidchromatography (such as reverse-phase chromatography, size-exclusionchromatography), gel electrophoresis, and translational assays to assessthe quality and purity of nucleic acid production. RNA preparationquality can also be determined using Bioanalyzer chip-basedelectrophoresis system. In vitro efficacy can be analyzed by, e.g.,transfecting RNA transcript into a human cell line, e.g., HeLA, PBMC, BJFibroblasts, Hek 293). Protein expression of the polypeptide of interestcan be quantified using methods such as Enzyme-Linked ImmunosorbantAssay (ELISA), western blot, or flow cytometry.

A variety of methods have been used to detect and/or quantitate dsRNAusing dsRNA-specific antibodies. These include ELISA, for example,sandwich ELISA (Schonborn et al. (1991) Nucleic Acids Res 19:2993-3000),dot-blots (for quantitation, specificity testing) (Kariko et al. (2011)Nucleic Acids Res 39:e142), and immunoprecipitation/immunoblotting. Inexemplary aspects of the invention, contaminants may be recognized usingan ELISA. A K1/J2 or K2/J2 assay may be used to determine the abundanceof dsRNA contaminants in a sample. An exemplary ELISA is a sandwichELISA, as follows. Blocking: Microtiter plates are pre-coated withprotein, e.g., 0.4 μg/well protein A at 4° C. overnight. Free bindingsites are saturated with bovine serum albumin (BSA) (e.g., 2%) in buffer(e.g.) PBS and the plates are then washed with buffer (e.g., PBS) andstored at 4° C. The dsRNA-specific J2 monoclonal antibody (IgG2a) isimmobilized onto the protein A layer by incubation of hybridomasupernatant (e.g. 100 μl per well at 4° C. overnight. The plates arewashed multiple times with buffer, e.g., PBS plus Tween 20 (e.g., 0.5%(v/v) Tween 20) and nucleic acid samples are added in buffer (e.g., TEbuffer, 37° C., 2 h). After washing as above, bound nucleic acid (i.e.,J2 antigens) are identified by successive incubation with the dilutedhybridoma supernatant (e.g., 1:2) of the dsRNA-specific K2 IgMmonoclonal antibody followed by an alkaline phosphatase conjugatedsecondary antibody (e.g., 1:5000 diluted goat anti-mouse IgM). Bothincubation steps are carried out al 37° C. for ˜1-2 h. Washing,substrate incubation and reading of absorption are performed accordingto art recognized methods.

A similar assay using dot blots is described by Kariko et al., Nuc.Acids Res. 2011; 39(21):e142. The assay is performed by blotting RNA(200 ng) onto super-charged Nytran membranes, where it is dried andblocked with 5% non-fat dried milk in TBS-T buffer (50 mM Tris-HCl, 150mM NaCl, 0.05% Tween-20, pH 7.4). The sample is then incubated with adsRNA-specific K1 or J2 monoclonal antibody (IgG) for one hour. Themembranes may be washed with TBS-T and incubated with an HRP-conjugatedanti-goat polyclonal antibody, for example. The membranes are washedagain, and the signal is detected using TMB. The signal is developedwith the addition of TMB. The assay is useful for detecting dsRNAduplexes greater than 40 base pairs in length.

Cytokine assays may also be used to detect RNA contaminants. Numerouscytokine assays are known in the art. The assays may test for theinduction of any cytokine associated with impure IVT products. Thesecytokines include for instance, interleukins (IL), interference (IFN)alphas, beta, and gamma, and TNF. In one embodiment, an IFN-β cell-basedassay may be used. Its results have been shown to correlate with thepresence of RNaseIII substrate as detected by LC-MS. Other cell-basedcytokine assays, such as for example IL or multiplex cytokine assays maybe used.

In exemplary BJF IFN-beta and hEPO expression assays BJ Fibroblastscells (ATCC) are seeded in a cell culture plate. Approximately 24 hoursafter seeding, the cells are transfected with mRNA using lipofectamineor other delivery agent. After transfection, supernatants are collectedand IFN-beta expression is measured using the human IFN-beta ELISA kit,High Sensitivity per manufacturer's instructions (PBL Assay Science).Briefly, human IFN-β is measured in cell supernatants by indirect enzymelinked immunosorbent assay (ELISA). Pre-coated plates are incubated withcell supernatants then washed to remove non-specifically bound material.IFN-β expression is analyzed by incubating the wells with anti-IFN-βantibody followed by a secondary antibody conjugated to horseradishperoxidase (HRP). Tetramethylbenzidine (TMB) is the HRP substrate usedfor detection. Human Epo levels are measured using Epo Human ELISA Kit(Thermo Fisher).

In exemplary Luminex assays, serum from mice are collected to assess thecytokine levels using Luminex screening assay technology (R&D Systems).Briefly, analyte-specific antibodies are pre-coated onto color-codedbeads. Beads, standards, and samples are pipetted into wells and theimmobilized antibodies bind the analytes of interest. After washing awayany unbound substances, a biotinylated antibody cocktail specific to theanalytes of interest is added to each well. Following a wash to removeany unbound biotinylated antibody, Streptavidin-Phycoerythrin conjugate(Streptavidin-PE), which binds to the biotinylated detection antibodies,is added to each well. A final wash removes unbound Streptavidin-PE andthe beads are resuspended in buffer and read using a Luminex analyzer(R&D Systems). A first laser is bead-specific and determines whichanalyte is being detected. A second laser determines the magnitude ofthe PE-derived signal, which is in direct proportion to the amount ofanalyte bound.

Surrogate Assays for Purity

In exemplary aspects of the invention, it may be desirable to determinepurity by use of a surrogate assay that is amenable to highlyqualitative and/or quantitative detection of products and/or impurities.Accordingly, the invention contemplates determination of purity of anRNA composition, e.g., an IVT RNA, produced by a certain IVT method, bydetermining purity of a surrogate RNA (e.g., a model RNA) produced byidentical conditions. In this manner, purity can be determinedindirectly via highly qualitative and/or quantitative detection methodsin a surrogate system, this purity determination correlating to purityof an IVT RNA produced in a production system. Furthermore, areconstitution or surrogate type assay may be used to determine theamount and identity of contaminants in a given RNA preparationindirectly. It may be difficult in some instances to detect low levelsof contaminants or contaminants having similar structural properties tothe RNA transcripts. Reconstitution systems can be used to testbiological activity such as immune stimulatory activity e.g. cytokineactivity associated with contaminants by adding back the putativecontaminants which are missing from the RNA preparations of theinvention in comparison to biological activity by RNA compositionsproduced by the traditional IVT methods. The reconstitution of the pureRNA preparations of the invention with putative contaminants candemonstrate the lack of the contaminants in the pure RNA preparations.

Additionally, model RNAs (surrogate mRNAs) may be used. Under the sameIVT conditions used to produce the IVT RNA, a model RNA from a DNAtemplate encoding the model RNA is produced. A model RNA or surrogatemRNA, as used herein, refers to a noncoding RNA transcript consisting ofonly the 5′ UTR, 3′ UTR, and polyA tail. A short model RNA may also beused. A short model RNA is a shorter version of model RNA (only consistsof 5′UTR and a shorter polyA tail (A20)). The amount of reversecomplement transcription product or cytokine-inducing species in thecomposition is determined by LC-MS or other analytical methods, as theamount of model RNA indicates the amount of reverse complementtranscription product or cytokine-inducing species in the composition.The amount and nature of the contaminants detected in the assaycorrelates and predicts the amount and nature of the contaminants thatwould be obtained using the identical IVT reaction conditions togenerate full-length mRNAs.

The RNA preparation of the invention is a high quality preparation. Insome embodiments the RNA preparation resulting directly from an IVTprocess may be used directly as a research reagent or a diagnostic ortherapeutic reagent without further purification. In some embodimentsthe RNA preparation may be subjected to one or more purification steps.For instance, the RNA preparation may be purified from truncated RNA,DNA template, and residual enzymes using oligo dT chromatography. Anexemplary oligo dT chromatography assay involves packing 20 merpolythymidine Sepharose (3 ml) in a 5 mL SPE column on a solid phaseextraction vacuum manifold. The RNA transcript is applied to column,followed by washing and elution. The oligo dT purified RNA transcript isdiafiltered into water and concentrated to 1.22 mg/mL using 100 kDa MWCOAmicon spin filters (EMD Millipore). The RNA is recovered and theconcentration may be determined using Bioanalyzer gel electrophoresis.

The analysis of the RNA preparation to determine purity and quality ofthe sample can be performed before or after capping. Alternatively,analysis can be performed before or after poly A capture based affinitypurification. In another embodiment, analysis can be performed before orafter optional additional purification steps, e.g., anion exchangechromatography and the like.

Mass spectrometry encompasses a broad range of techniques foridentifying and characterizing compounds in mixtures. Different types ofmass spectrometry-based approaches may be used to analyze a sample todetermine its composition. Mass spectrometry analysis involvesconverting a sample being analyzed into multiple ions by an ionizationprocess. Each of the resulting ions, when placed in a force field, movesin the field along a trajectory such that its acceleration is inverselyproportional to its mass-to-charge ratio. A mass spectrum of a moleculeis thus produced that displays a plot of relative abundances ofprecursor ions versus their mass-to-charge ratios. When a subsequentstage of mass spectrometry, such as tandem mass spectrometry, is used tofurther analyze the sample by subjecting precursor ions to higherenergy, each precursor ion may undergo disassociation into fragmentsreferred to as product ions. Resulting fragments can be used to provideinformation concerning the nature and the structure of their precursormolecule.

MALDI-TOF (matrix-assisted laser desorption ionization time of flight)mass spectrometry provides for the spectrometric determination of themass of poorly ionizing or easily-fragmented analytes of low volatilityby embedding them in a matrix of light-absorbing material and measuringthe weight of the molecule as it is ionized and caused to fly byvolatilization. Combinations of electric and magnetic fields are appliedon the sample to cause the ionized material to move depending on theindividual mass and charge of the molecule. U.S. Pat. No. 6,043,031,issued to Koster et al., describes an exemplary method for identifyingsingle-base mutations within DNA using MALDI-TOF and other methods ofmass spectrometry.

HPLC (high performance liquid chromatography) is used for the analyticalseparation of bio-polymers, based on properties of the bio-polymers.HPLC can be used to separate nucleic acid sequences based on sizecharge, and base composition. A nucleic acid sequence having one basepair difference from another nucleic acid can be separated using HPLC.Thus, nucleic acid samples, which are identical except for a singlenucleotide may be differentially separated using HPLC, to identify thepresence or absence of a particular nucleic acid fragments. Preferablythe HPLC is HPLC-UV.

In some embodiments, the RNA may be purified without using a dsRNasestep. For example, RNaseIII may not be used. The composition may beproduced by a process that does not use reversed-phase chromatographypurification step. In one embodiment, the RNA composition may beproduced without using high-performance lipid chromatography (HPLC)purification. Thus, the composition is free of residual organic reagentsor contaminants associated with traditional purification processes.

In some instances, the methods of the invention are used to determinethe purity of an RNA sample. The term “pure” as used herein refers tomaterial that has only the target nucleic acid active agents such thatthe presence of unrelated nucleic acids is reduced or eliminated, i.e.,impurities or contaminants, including RNA fragments. For example, apurified RNA sample includes one or more target or test nucleic acidsbut is preferably substantially free of other nucleic acids detectableby methods described. As used herein, the term “substantially free” isused operationally, in the context of analytical testing of thematerial. Preferably, purified material is substantially free of one ormore impurities or contaminants including the reverse complementtranscription products and/or cytokine-inducing RNA contaminantdescribed herein and for instance is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, or 97% pure; more preferably, at least 98% pure, and morepreferably still at least 99% pure. In some embodiments a pure RNAsample is comprised of 100°/% of the target or test RNAs and includes noother RNA.

In some embodiments, capillary electrophoresis (CE) is used to separatethe RNA. An electric field is applied to the sample so that it runsthrough an electrolyte solution, such as an aqueous buffer solution, toa destination vial via a capillary. The analytes migrate differentlybased on electrophoretic mobility and are detected at the outlet end ofthe capillary. The output data is recorded and then displayed as anelectropherogram. It can be used in conjunction with mass spectrometryto determine the identity of sample components. The capillaryelectrophoresis system is fully automated in the FRAGMENT ANALYZER™,which can detect differences as small as three base pairs.

In some embodiments, a fragment analyzer (FA) may be used to quantifyand purify the RNA. The fragment analyzer automates capillaryelectrophoresis and HPLC.

In some embodiments, the RNA molecules are mRNA molecules. As usedherein, the term “messenger RNA” (mRNA) refers to any polynucleotidewhich encodes at least one peptide or polypeptide of interest and whichis capable of being translated to produce the encoded peptidepolypeptide of interest in vitro, in vivo, in situ or ex vivo. An mRNAhas been transcribed from a DNA sequence by an RNA polymerase enzyme,and interacts with a ribosome synthesize genetic information encoded byDNA. Generally, mRNA is classified into two sub-classes: pre-mRNA andmature mRNA. Precursor mRNA (pre-mRNA) is mRNA that has been transcribedby RNA polymerase but has not undergone any post-transcriptionalprocessing (e.g., 5′capping, splicing, editing, and polyadenylation).Mature mRNA has been modified via post-transcriptional processing (e.g.,spliced to remove introns and polyadenylated) and is capable ofinteracting with ribosomes to perform protein synthesis. mRNA can beisolated from tissues or cells by a variety of methods. For example, atotal RNA extraction can be performed on cells or a cell lysate and theresulting extracted total RNA can be purified (e.g., on a columncomprising oligo-dT beads) to obtain extracted mRNA.

Alternatively, mRNA can be synthesized in a cell-free environment, forexample by in vitro transcription (IVT). An “in vitro transcriptiontemplate” as used herein, refers to deoxyribonucleic acid (DNA) suitablefor use in an IVT reaction for the production of messenger RNA (mRNA).In some embodiments, an IVT template encodes a 5′ untranslated region,contains an open reading frame, and encodes a 3′ untranslated region anda polyA tail. The particular nucleotide sequence composition and lengthof an IVT template will depend on the mRNA of interest encoded by thetemplate.

A “5′ untranslated region (UTR)” refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode aprotein or peptide.

A “3′ untranslated region (UTR)” refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a protein or peptide.

An “open reading frame” is a continuous stretch of DNA beginning with astart codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) and encodes a protein or peptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400,450, 500, 550, or 600 adenosine monophosphates. In some embodiments, apolyA tail contains 50 to 250 adenosine monophosphates. In a relevantbiological setting (e.g., in cells, in vivo, etc.) the poly(A) tailfunctions to protect mRNA from enzymatic degradation, e.g., in thecytoplasm, and aids in transcription termination, export of the mRNAfrom the nucleus, and translation.

Thus, the polynucleotide may in some embodiments comprise (a) a firstregion of linked nucleotides encoding a polypeptide of interest; (b) afirst terminal region located 5′ relative to said first regioncomprising a 5′ untranslated region (UTR); (c) a second terminal regionlocated 3′ relative to said first region; and (d) a tailing region. Theterms poly nucleotide and nucleic acid are used interchangeably herein.

In some embodiments, the polynucleotide includes from about 1 to about3,000, 10 to about 3,000, 20 to about 3,000, 30 to about 3,000, 40 toabout 3,000, 50 to about 3,000, 100 to 20 about 3,000, 200 to about3,000 nucleotides (e.g., from 200 to 500, from 200 to 1,000, from 200 to1,500, from 200 to 3,000, from 500 to 1,000, from 500 to 1,500, from 500to 2,000, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000,from 1,000 to 3,000, from 1,500 to 3,000, and from 2,000 to 3,000).

IVT RNA may function as RNA but are distinguished from wild-type RNA intheir functional and/or structural design features which serve toovercome existing problems of effective polypeptide production usingnucleic-acid based therapeutics. For example, IVT RNA may bestructurally modified or chemically modified. As used herein, a“structural” modification is one in which two or more linked nucleotidesare inserted, deleted, duplicated, inverted or randomized in apolynucleotide without significant chemical modification to thenucleotides themselves. Because chemical bonds will necessarily bebroken and reformed to effect a structural modification, structuralmodifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to “AT-5meC-G”. The samepolynucleotide may be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

cDNA encoding the polynucleotides described herein may be transcribedusing an in vitro transcription (IVT) system. The system typicallycomprises a transcription buffer, nucleotide triphosphates (NTPs), anRNase inhibitor and a polymerase. The NTPs may be manufactured in house,may be selected from a supplier, or may be synthesized as describedherein. The NTPs may be selected from, but are not limited to, thosedescribed herein including natural and unnatural (modified) NTPs. Thepolymerase may be selected from, but is not limited to, T7 RNApolymerase, T3 RNA polymerase and mutant polymerases such as, but notlimited to, polymerases able to incorporate polynucleotides (e.g.,modified nucleic acids).

Chemically-Modified RNAs

Thus, in an exemplary aspect, polynucleotides of the invention mayinclude at least one chemical modification. The polynucleotides caninclude various substitutions and/or insertions from native or naturallyoccurring polynucleotides. As used herein in a polynucleotide, the terms“chemical modification” or, as appropriate, “chemically modified” referto modification with respect to adenosine (A), guanosine (G), uridine(U), thymidine (T) or cytidine (C) ribo- or deoxyribnucleosides in oneor more of their position, pattern, percent or population. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal RNA cap moieties.

The modifications may be various distinct modifications. In someembodiments, the regions may contain one, two, or more (optionallydifferent) nucleoside or nucleotide modifications. In some embodiments,a modified polynucleotide, introduced to a cell may exhibit reduceddegradation in the cell, as compared to an unmodified polynucleotide.

Modifications of the polynucleotides include, but are not limited tothose listed in detail below. The polynucleotide may comprisemodifications which are naturally occurring, non-naturally occurring orthe polynucleotide can comprise both naturally and non-naturallyoccurring modifications.

The polynucleotides of the invention can include any usefulmodification, such as to the sugar, the nucleobase, or theinternucleotide linkage (e.g. to a linking phosphate/to a phosphodiesterlinkage/to the phosphodiester backbone). One or more atoms of apyrimidine nucleobase may be replaced or substituted with optionallysubstituted amino, optionally substituted thiol, optionally substitutedalkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). Incertain embodiments, modifications (e.g., one or more modifications) arepresent in each of the sugar and the internucleotide linkage.Modifications according to the present invention may be modifications ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additionalmodifications are described herein.

Non-natural modified nucleotides may be introduced to polynucleotidesduring synthesis or post-synthesis of the chains to achieve desiredfunctions or properties. The modifications may be on internucleotidelineage, the purine or pyrimidine bases, or sugar. The modification maybe introduced at the terminal of a chain or anywhere else in the chain;with chemical synthesis or with a polymerase enzyme. Any of the regionsof the polynucleotides may be chemically modified.

The present disclosure provides for polynucleotides comprised ofunmodified or modified nucleosides and nucleotides and combinationsthereof. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. The modified nucleotides may bysynthesized by any useful method, as described herein (e.g., chemically,enzymatically, or recombinantly to include one or more modified ornon-natural nucleotides). The polynucleotides may comprise a region orregions of linked nucleotides. Such regions may have variable backbonelinkages. The linkages may be standard phosphodiester linkages, in whichcase the polynucleotides would comprise regions of nucleotides. Anycombination of base/sugar or linker may be incorporated into thepolynucleotides of the invention.

In some embodiments, an RNA of the invention includes a combination ofone or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases.)

Modifications of the nucleic acids which are useful in the presentinvention include, but are not limited to those in the Table below.

Natu- rally Oc- Name Symbol Base curring 2-methylthio-N6-(cis- ms2i6A AYES hydroxyisopentenyl)adenosine 2-methylthio-N6-methyladenosine ms2m6AA YES 2-methylthio-N6-threonyl ms2t6A A YES carbamoyladenosineN6-glycinylcarbamoyladenosine g6A A YES N6-isopentenyladenosine i6A AYES N6-methyladenosine m6A A YES N6-threonylcarbamoyladenosine t6A A YES1,2′-O-dimethyladenosine m1Am A YES 1-methyladenosine m1A A YES2′-O-methyladenosine Am A YES 2′-O-ribosyladenosine (phosphate) Ar(p) AYES 2-methyladenosine m2A A YES 2-methylthio-N6 isopentenyladenosinems2i6A A YES 2-methylthio-N6-hydroxynorvalyl ms2hn6A A YEScarbamoyladenosine 2′-O-methyladenosine m6A A YES 2′-O-ribosyladenosine(phosphate) Ar(p) A YES Isopentenyladenosine Iga A YESN6-(cis-hydroxyisopentenyl)adenosine io6A A YESN6,2′-O-dimethyladenosine m6Am A YES N6,2′-O-dimethyladenosine m6Am AYES N6,N6,2′-O-trimethyladenosine m62Am A YES N6,N6-dimethyladenosinem62A A YES N6-acetyladenosine ac6A A YES N6- hn6A A YEShydroxynorvalylcarbamoyladenosine N6-methyl-N6- m6t6A A YESthreonylcarbamoyladenosine 2-methyladenosine m2A A YES2-methylthio-N6-isopentenyladenosine ms2i6A A YES 7-deaza-adenosine — ANO N1-methyl-adenosine — A NO N6, N6 (dimethyl)adenine — A NON6-cis-hydroxy-isopentenyl-adenosine — A NO α-thio-adenosine — A NO 2(amino)adenine — A NO 2 (aminopropyl)adenine — A NO 2 (methylthio) N6 —A NO (isopentenyl)adenine 2-(alkyl)adenine — A NO 2-(aminoalkyl)adenine— A NO 2-(aminopropyl)adenine — A NO 2-(halo)adenine — A NO2-(halo)adenine — A NO 2-(propyl)adenine — A NO 2′-Amino-2′-deoxy-ATP —A NO 2′-Azido-2′-deoxy-ATP — A NO 2′-Deoxy-2′-a-aminoadenosine TP — A NO2′-Deoxy-2′-a-azidoadenosine TP — A NO 6 (alkyl)adenine — A NO 6(methyl)adenine — A NO 6-(alkyl)adenine — A NO 6-(methyl)adenine — A NO7 (deaza)adenine — A NO 8 (alkenyl)adenine — A NO 8 (alkynyl)adenine — ANO 8 (amino)adenine — A NO 8 (thioalkyl)adenine — A NO8-(alkenyl)adenine — A NO 8-(alkyl)adenine — A NO 8-(alkynyl)adenine — ANO 8-(amino)adenine — A NO 8-(halo)adenine — A NO 8-(hydroxyl)adenine —A NO 8-(thioalkyl)adenine — A NO 8-(thiol)adenine — A NO8-azido-adenosine — A NO aza adenine — A NO deaza adenine — A NO N6(methyl)adenine — A NO N6-(isopentyl)adenine — A NO7-deaza-8-aza-adenosine — A NO 7-methyladenine — A NO 1-DeazaadenosineTP — A NO 2′Fluoro-N6-Bz-deoxyadenosine TP — A NO 2′-OMe-2-Amino-ATP — ANO 2′O-methyl-N6-Bz-deoxyadenosine — A NO TP 2′-a-Ethynyladenosine TP —A NO 2-aminoadenine — A NO 2-Aminoadenosine TP — A NO 2-Amino-ATP — A NO2′-a-Trifluoromethyladenosine TP — A NO 2-Azidoadenosine TP — A NO2′-b-Ethynyladenosine TP — A NO 2-Bromoadenosine TP — A NO2′-b-Trifluoromethyladenosine TP — A NO 2-Chloroadenosine TP — A NO2′-Deoxy-2′,2′-difluoroadenosine TP — A NO2′-Deoxy-2′-a-mercaptoadenosine TP — A NO2′-Deoxy-2′-a-thiomethoxyadenosine — A NO TP2′-Deoxy-2′-b-aminoadenosine TP — A NO 2′-Deoxy-2′-b-azidoadenosine TP —A NO 2′-Deoxy-2′-b-bromoadenosine TP — A NO2′-Deoxy-2′-b-chloroadenosine TP — A NO 2′-Deoxy-2′-b-fluoroadenosine TP— A NO 2′-Deoxy-2′-b-iodoadenosine TP — A NO2′-Deoxy-2′-b-mercaptoadenosine TP — A NO2′-Deoxy-2′-b-thiomethoxyadenosine — A NO TP 2-Fluoroadenosine TP — A NO2-Iodoadenosine TP — A NO 2-Mercaptoadenosine TP — A NO2-methoxy-adenine — A NO 2-methylthio-adenine — A NO2-Trifluoromethyladenosine TP — A NO 3-Deaza-3-bromoadenosine TP — A NO3-Deaza-3-chloroadenosine TP — A NO 3-Deaza-3-fluoroadenosine TP — A NO3-Deaza-3-iodoadenosine TP — A NO 3-Deazaadenosine TP — A NO4′-Azidoadenosine TP — A NO 4′-Carbocyclic adenosine TP — A NO4′-Ethynyladenosine TP — A NO 5′-Homo-adenosine TP — A NO 8-Aza-ATP — ANO 8-bromo-adenosine TP — A NO 8-Trifluoromethyladenosine TP — A NO9-Deazaadenosine TP — A NO 2-aminopurine — A/G NO7-deaza-2,6-diaminopurine — A/G NO 7-deaza-8-aza-2,6-diaminopurine — A/GNO 7-deaza-8-aza-2-aminopurine — A/G NO 2,6-diaminopurine — A/G NO7-deaza-8-aza-adenine, 7-deaza-2- — A/G NO aminopurine 2-thiocytidines2C C YES 3-methylcytidine m3C C YES 5-formylcytidine f5C C YES5-hydroxymethylcytidine hm5C C YES 5-methylcytidine m5C C YESN4-acetylcytidine ac4C C YES 2′-O-methylcytidine Cm C YES2′-O-methylcytidine Cm C YES 5,2′-O-dimethylcytidine m5 Cm C YES5-formyl-2′-O-methylcytidine f5Cm C YES Lysidine k2C C YESN4,2′-O-dimethylcytidine m4Cm C YES N4-acetyl-2′-O-methylcytidine ac4CmC YES N4-methylcytidine m4C C YES N4,N4-Dimethyl-2′-OMe-Cytidine TP — CYES 4-methylcytidine — C NO 5-aza-cytidine — C NO Pseudo-iso-cytidine —C NO pyrrolo-cytidine — C NO α-thio-cytidine — C NO 2-(thio)cytosine — CNO 2′-Amino-2′-deoxy-CTP — C NO 2′-Azido-2′-deoxy-CTP — C NO2′-Deoxy-2′-a-aminocytidine TP — C NO 2′-Deoxy-2′-a-azidocytidine TP — CNO 3 (deaza) 5 (aza)cytosine — C NO 3 (methyl)cytosine — C NO3-(alkyl)cytosine — C NO 3-(deaza) 5 (aza)cytosine — C NO3-(methyl)cytidine — C NO 4,2′-O-dimethylcytidine — C NO 5(halo)cytosine — C NO 5 (methyl)cytosine — C NO 5 (propynyl)cytosine — CNO 5 (trifluoromethyl)cytosine — C NO 5-(alkyl)cytosine — C NO5-(alkynyl)cytosine — C NO 5-(halo)cytosine — C NO 5-(propynyl)cytosine— C NO 5-(trifluoromethyl)cytosine — C NO 5-bromo-cytidine — C NO5-iodo-cytidine — C NO 5-propynyl cytosine — C NO 6-(azo)cytosine — C NO6-aza-cytidine — C NO aza cytosine — C NO deaza cytosine — C NO N4(acetyl)cytosine — C NO 1-methyl-1-deaza-pseudoisocytidine — C NO1-methyl-pseudoisocytidine — C NO 2-methoxy-5-methyl-cytidine — C NO2-methoxy-cytidine — C NO 2-thio-5-methyl-cytidine — C NO4-methoxy-1-methyl- — C NO pseudoisocytidine 4-methoxy-pseudoisocytidine— C NO 4-thio-1-methyl-1-deaza- — C NO pseudoisocytidine4-thio-1-methyl-pseudoisocytidine — C NO 4-thio-pseudoisocytidine — C NO5-aza-zebularine — C NO 5-methyl-zebularine — C NOpyrrolo-pseudoisocytidine — C NO Zebularine — C NO(E)-5-(2-Bromo-vinyl)cytidine TP — C NO 2,2′-anhydro-cytidine TP — C NOhydrochloride 2′Fluor-N4-Bz-cytidine TP — C NO2′Fluoro-N4-Acetyl-cytidine TP — C NO 2′-O-Methyl-N4-Acetyl-cytidine TP— C NO 2′O-methyl-N4-Bz-cytidine TP — C NO 2′-a-Ethynylcytidine TP — CNO 2′-a-Trifluoromethylcytidine TP — C NO 2′-b-Ethynylcytidine TP — C NO2′-b-Trifluoromethylcytidine TP — C NO 2′-Deoxy-2′,2′-difluorocytidineTP — C NO 2′-Deoxy-2′-a-mercaptocytidine TP — C NO2′-Deoxy-2′-a-thiomethoxycytidine TP — C NO 2′-Deoxy-2′-b-aminocytidineTP — C NO 2′-Deoxy-2′-b-azidocytidine TP — C NO2′-Deoxy-2′-b-bromocytidine TP — C NO 2′-Deoxy-2′-b-chlorocytidine TP —C NO 2′-Deoxy-2′-b-fluorocytidine TP — C NO 2′-Deoxy-2′-b-iodocytidineTP — C NO 2′-Deoxy-2′-b-mercaptocytidine TP — C NO2′-Deoxy-2′-b-thiomethoxycytidine TP — C NO2′-O-Methyl-5-(1-propynyl)cytidine — C NO TP 3′-Ethynylcytidine TP — CNO 4′-Azidocytidine TP — C NO 4′-Carbocyclic cytidine TP — C NO4′-Ethynylcytidine TP — C NO 5-(1-Propynyl)ara-cytidine TP — C NO5-(2-Chloro-phenyl)-2-thiocytidine TP — C NO5-(4-Amino-phenyl)-2-thiocytidine TP — C NO 5-Aminoallyl-CTP — C NO5-Cyanocytidine TP — C NO 5-Ethynylara-cytidine TP — C NO5-Ethynylcytidine TP — C NO 5′-Homo-cytidine TP — C NO 5-MethoxycytidineTP — C NO 5-Trifluoromethyl-Cytidine TP — C NO N4-Amino-cytidine TP — CNO N4-Benzoyl-cytidine TP — C NO Pseudoisocytidine — C NO7-methylguanosine m7G G YES N2,2′-O-dimethylguanosine m2Gm G YESN2-methylguanosine m2G G YES Wyosine imG G YES 1,2′-O-dimethylguanosinem1Gm G YES 1-methylguanosine m1G G YES 2′-O-methylguanosine Gm G YES2′-O-ribosylguanosine (phosphate) Gr(p) G YES 2′-O-methylguanosine Gm GYES 2′-O-ribosylguanosine (phosphate) Gr(p) G YES7-aminomethyl-7-deazaguanosine preQ1 G YES 7-cyano-7-deazaguanosinepreQ0 G YES Archaeosine G+ G YES Methylwyosine mimG G YESN2,7-dimethylguanosine m2,7G G YES N2,N2,2′-O-trimethylguanosine m22Gm GYES N2,N2,7-trimethylguanosine m2,2,7G G YES N2,N2-dimethylguanosinem22G G YES N2,7,2′-O-trimethylguanosine m2,7Gm G YES 6-thio-guanosine —G NO 7-deaza-guanosine — G NO 8-oxo-guanosine — G NO N1-methyl-guanosine— G NO α-thio-guanosine — G NO 2 (propyl)guanine — G NO 2-(alkyl)guanine— G NO 2′-Amino-2′-deoxy-GTP — G NO 2′-Azido-2′-deoxy-GTP — G NO2′-Deoxy-2′-a-aminoguanosine TP — G NO 2′-Deoxy-2′-a-azidoguanosine TP —G NO 6 (methyl)guanine — G NO 6-(alkyl)guanine — G NO 6-(methyl)guanine— G NO 6-methyl-guanosine — G NO 7 (alkyl)guanine — G NO 7(deaza)guanine — G NO 7 (methyl)guanine — G NO 7-(alkyl)guanine — G NO7-(deaza)guanine — G NO 7-(methyl)guanine — G NO 8 (alkyl)guanine — G NO8 (alkynyl)guanine — G NO 8 (halo)guanine — G NO 8 (thioalkyl)guanine —G NO 8-(alkenyl)guanine — G NO 8-(alkyl)guanine — G NO8-(alkynyl)guanine — G NO 8-(amino)guanine — G NO 8-(halo)guanine — G NO8-(hydroxyl)guanine — G NO 8-(thioalkyl)guanine — G NO 8-(thiol)guanine— G NO aza guanine — G NO deaza guanine — G NO N (methyl)guanine — G NON-(methyl)guanine — G NO 1-methyl-6-thio-guanosine — G NO6-methoxy-guanosine — G NO 6-thio-7-deaza-8-aza-guanosine — G NO6-thio-7-deaza-guanosine — G NO 6-thio-7-methyl-guanosine — G NO7-deaza-8-aza-guanosine — G NO 7-methyl-8-oxo-guanosine — G NON2,N2-dimethyl-6-thio-guanosine — G NO N2-methyl-6-thio-guanosine — G NO1-Me-GTP — G NO 2′Fluoro-N2-isobutyl-guanosine TP — G NO2′O-methyl-N2-isobutyl-guanosine TP — G NO 2′-a-Ethynylguanosine TP — GNO 2′-a-Trifluoromethylguanosine TP — G NO 2′-b-Ethynylguanosine TP — GNO 2′-b-Trifluoromethylguanosine TP — G NO2′-Deoxy-2′,2′-difluoroguanosine TP — G NO2′-Deoxy-2′-a-mercaptoguanosine TP — G NO2′-Deoxy-2′-a-thiomethoxyguanosine — G NO TP2′-Deoxy-2′-b-aminoguanosine TP — G NO 2′-Deoxy-2′-b-azidoguanosine TP —G NO 2′-Deoxy-2′-b-bromoguanosine TP — G NO2′-Deoxy-2′-b-chloroguanosine TP — G NO 2′-Deoxy-2′-b-fluoroguanosine TP— G NO 2′-Deoxy-2′-b-iodoguanosine TP — G NO2′-Deoxy-2′-b-mercaptoguanosine TP — G NO2′-Deoxy-2′-b-thiomethoxyguanosine — G NO TP 4′-Azidoguanosine TP — G NO4′-Carbocyclic guanosine TP — G NO 4′-Ethynylguanosine TP — G NO5′-Homo-guanosine TP — G NO 8-bromo-guanosine TP — G NO 9-DeazaguanosineTP — G NO N2-isobutyl-guanosine TP — G NO 1-methylinosine m1I I YESInosine I I YES 1,2′-O-dimethylinosine m1Im I YES 2′-O-methylinosine ImI YES 7-methylinosine I NO 2′-O-methylinosine Im I YES Epoxyqueuosine oQQ YES galactosyl-queuosine galQ Q YES Mannosylqueuosine manQ Q YESQueuosine Q Q YES allyamino-thymidine — T NO aza thymidine — T NO deazathymidine — T NO deoxy-thymidine — T NO 2′-O-methyluridine — U YES2-thiouridine s2U U YES 3-methyluridine m3U U YES 5-carboxymethyluridinecm5U U YES 5-hydroxyuridine ho5U U YES 5-methyluridine m5U U YES5-taurinomethyl-2-thiouridine τm5s2U U YES 5-taurinomethyluridine τm5U UYES Dihydrouridine d U YES Pseudouridine Ψ U YES(3-(3-amino-3-carboxypropyl)uridine acp3U U YES 1-methyl-3-(3-amino-5-m1acp3Ψ U YES carboxypropyl)pseudouridine 1-methylpseduouridine m1Ψ UYES 1-methyl-pseudouridine — U YES 2′-O-methyluridine Um U YES2′-O-methylpseudouridine Ψm U YES 2′-O-methyluridine Um U YES2-thio-2′-O-methyluridine s2Um U YES 3-(3-amino-3-carboxypropyl)uridineacp3U U YES 3,2′-O-dimethyluridine m3Um U YES 3-Methyl-pseudo-Uridine TP— U YES 4-thiouridine s4U U YES 5-(carboxyhydroxymethyl)uridine chm5U UYES 5-(carboxyhydroxymethyl)uridine mchm5U U YES methyl ester5,2′-O-dimethyluridine m5Um U YES 5,6-dihydro-uridine — U YES5-aminomethyl-2-thiouridine nm5s2U U YES 5-carbamoylmethyl-2′-O- ncm5UmU YES methyluridine 5-carbamoylmethyluridine ncm5U U YES5-carboxyhydroxymethyluridine — U YES 5-carboxyhydroxymethyluridine — UYES methyl ester 5-carboxymethylaminomethyl-2′-O- cmnm5Um U YESmethyluridine 5-carboxymethylaminomethyl-2- cmnm5s2U U YES thiouridine5-carboxymethylaminomethyl-2- — U YES thiouridine5-carboxymethylaminomethyluridine cmnm5U U YES5-carboxymethylaminomethyluridine — U YES 5-Carbamoylmethyluridine TP —U YES 5-methoxycarbonylmethyl-2′-O- mcm5Um U YES methyluridine5-methoxycarbonylmethyl-2- mcm5s2U U YES thiouridine5-methoxycarbonylmethyluridine mcm5U U YES 5-methoxyuridine mo5U U YES5-methyl-2-thiouridine m5s2U U YES 5-methylaminomethyl-2-selenouridinemnm5se2U U YES 5-methylaminomethyl-2-thiouridine mnm5s2U U YES5-methylaminomethyluridine mnm5U U YES 5-Methyldihydrouridine — U YES5-Oxyacetic acid- Uridine TP — U YES 5-Oxyacetic acid-methylester-Uridine — U YES TP N1-methyl-pseudo-uridine — U YES uridine5-oxyacetic acid cmo5U U YES uridine 5-oxyacetic acid methyl estermcmo5U U YES 3-(3-Amino-3-carboxypropyl)-Uridine — U YES TP5-(iso-Pentenylaminomethyl)-2- — U YES thiouridine TP5-(iso-Pentenylaminomethyl)-2′-O- — U YES methyluridine TP5-(iso-Pentenylaminomethyl)uridine — U YES TP 5-propynyl uracil — U NOα-thio-uridine — U NO 1 (aminoalkylamino- — U NOcarbonylethylenyl)-2(thio)- pseudouracil 1 — U NO(aminoalkylaminocarbonylethylenyl)- 2,4-(dithio)pseudouracil 1 — U NO(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil 1 — U NO(aminoalkylaminocarbonylethylenyl)- pseudouracil 1(aminocarbonylethylenyl)-2(thio)- — U NO pseudouracil 1(aminocarbonylethylenyl)-2,4- — U NO (dithio)pseudouracil 1(aminocarbonylethylenyl)-4 — U NO (thio)pseudouracil 1(aminocarbonylethylenyl)- — U NO pseudouracil 1 substituted2(thio)-pseudouracil — U NO 1 substituted 2,4-(dithio)pseudouracil — UNO 1 substituted 4 (thio)pseudouracil — U NO 1 substituted pseudouracil— U NO 1-(aminoalkylamino- — U NO carbonylethylenyl)-2-(thio)-pseudouracil 1-Methyl-3-(3-amino-3- — U NO carboxypropyl) pseudouridineTP 1-Methyl-3-(3-amino-3- — U NO carboxypropyl)pseudo-UTP1-Methyl-pseudo-UTP — U NO 2 (thio)pseudouracil — U NO 2′ deoxy uridine— U NO 2′ fluorouridine — U NO 2-(thio)uracil — U NO2,4-(dithio)psuedouracil — U NO 2′ methyl, 2′amino, 2′azido, 2′fluro- —U NO guanosine 2′-Amino-2′-deoxy-UTP — U NO 2′-Azido-2′-deoxy-UTP — U NO2′-Azido-deoxyuridine TP — U NO 2′-O-methylpseudouridine — U NO 2′ deoxyuridine 2′ dU U NO 2′ fluorouridine — U NO 2′-Deoxy-2′-a-aminouridine TP— U NO 2′-Deoxy-2′-a-azidouridine TP — U NO 2-methylpseudouridine m3Ψ UNO 3 (3 amino-3 carboxypropyl)uracil — U NO 4 (thio)pseudouracil — U NO4-(thio)pseudouracil — U NO 4-(thio)uracil — U NO 4-thiouracil — U NO 5(1,3-diazole-1-alkyl)uracil — U NO 5 (2-aminopropyl)uracil — U NO 5(aminoalkyl)uracil — U NO 5 (dimethylaminoalkyl)uracil — U NO 5(guanidiniumalkyl)uracil — U NO 5 (methoxycarbonylmethyl)-2- — U NO(thio)uracil 5 (methoxycarbonyl-methyl)uracil — U NO 5 (methyl) 2(thio)uracil — U NO 5 (methyl) 2,4 (dithio)uracil — U NO 5 (methyl) 4(thio)uracil — U NO 5 (methylaminomethyl)-2 (thio)uracil — U NO 5(methylaminomethyl)-2,4 — U NO (dithio)uracil 5 (methylaminomethyl)-4(thio)uracil — U NO 5 (propynyl)uracil — U NO 5 (trifiuoromethyl)uracil— U NO 5-(2-aminopropyl)uracil — U NO 5-(alkyl)-2-(thio)pseudouracil — UNO 5-(alkyl)-2,4 (dithio)pseudouracil — U NO 5-(alkyl)-4(thio)pseudouracil — U NO 5-(alkyl)pseudouracil — U NO 5-(alkyl)uracil —U NO 5-(alkynyl)uracil — U NO 5-(allylamino)uracil — U NO5-(cyanoalkyl)uracil — U NO 5-(dialkylaminoalkyl)uracil — U NO5-(dimethylaminoalkyl)uracil — U NO 5-(guanidiniumalkyl)uracil — U NO5-(halo)uracil — U NO 5-(1,3-diazole-1-alkyl)uracil — U NO5-(methoxy)uracil — U NO 5-(methoxycarbonylmethyl)-2- — U NO(thio)uracil 5-(methoxycarbonyl-methyl)uracil — U NO 5-(methyl)2(thio)uracil — U NO 5-(methyl) 2,4 (dithio)uracil — U NO 5-(methyl) 4(thio)uracil — U NO 5-(methyl)-2-(thio)pseudouracil — U NO5-(methyl)-2,4 (dithio)pseudouracil — U NO 5-(methyl)-4(thio)pseudouracil — U NO 5-(methyl)pseudouracil — U NO5-(methylaminomethyl)-2 (thio)uracil — U NO5-(methylaminomethyl)-2,4(dithio) — U NO uracil5-(methylaminomethyl)-4-(thio)uracil — U NO 5-(propynyl)uracil — U NO5-(trifluoromethyl)uracil — U NO 5-aminoallyl-uridine — U NO5-bromo-uridine — U NO 5-iodo-uridine — U NO 5-uracil — U NO 6(azo)uracil — U NO 6-(azo)uracil — U NO 6-aza-uridine — U NOallyamino-uracil — U NO aza uracil — U NO deaza uracil — U NO N3(methyl)uracil — U NO P seudo-UTP-1-2-ethanoic acid — U NO Pseudouracil— U NO 4-Thio-pseudo-UTP — U NO 1-carboxymethyl-pseudouridine — U NO1-methyl-1-deaza-pseudouridine — U NO 1-propynyl-uridine — U NO1-taurinomethyl-1-methyl-uridine — U NO 1-taurinomethyl-4-thio-uridine —U NO 1-taurinomethyl-pseudouridine — U NO 2-methoxy-4-thio-pseudouridine— U NO 2-thio-1-methyl-1-deaza-pseudouridine — U NO2-thio-1-methyl-pseudouridine — U NO 2-thio-5-aza-uridine — U NO2-thio-dihydropseudouridine — U NO 2-thio-dihydrouridine — U NO2-thio-pseudouridine — U NO 4-methoxy-2-thio-pseudouridine — U NO4-methoxy-pseudouridine — U NO 4-thio-1-methyl-pseudouridine — U NO4-thio-pseudouridine — U NO 5-aza-uridine — U NO Dihydropseudouridine —U NO (±)1-(2-Hydroxypropyl)pseudouridine — U NO TP (2R)-1-(2- — U NOHydroxypropyl)pseudouridine TP (2S)-1-(2- — U NOHydroxypropyl)pseudouridine TP (E)-5-(2-Bromo-vinyl)ara-uridine TP — UNO (E)-5-(2-Bromo-vinyl)uridine TP — U NO(Z)-5-(2-Bromo-vinyl)ara-uridine TP — U NO (Z)-5-(2-Bromo-vinyl)uridineTP — U NO 1-(2,2,2-Trifluoroethyl)-pseudo-UTP — U NO 1-(2,2,3,3,3- — UNO Pentafluoropropyl)pseudouridine TP 1-(2,2-Diethoxyethyl)pseudouridine— U NO TP 1-(2,4,6- — U NO Trimethylbenzyl)pseudouridine TP1-(2,4,6-Trimethyl-benzyl)pseudo- — U NO UTP1-(2,4,6-Trimethyl-phenyl)pseudo- — U NO UTP1-(2-Amino-2-carboxyethyl)pseudo- — U NO UTP 1-(2-Amino-ethyl)pseudo-UTP— U NO 1-(2-Hydroxyethyl)pseudouridine TP — U NO1-(2-Methoxyethyl)pseudouridine TP — U NO 1-(3,4-Bis- — U NOtrifluoromethoxybenzyl)pseudouridine TP 1-(3,4- — U NODimethoxybenzyl)pseudouridine TP 1-(3-Amino-3-carboxypropyl)pseudo- — UNO UTP 1-(3-Amino-propyl)pseudo-UTP — U NO 1-(3-Cyclopropyl-prop-2- — UNO ynyl)pseudouridine TP 1-(4-Amino-4-carboxybutyl)pseudo- — U NO UTP1-(4-Amino-benzyl)pseudo-UTP — U NO 1-(4-Amino-butyl)pseudo-UTP — U NO1-(4-Amino-phenyl)pseudo-UTP — U NO 1-(4-Azidobenzyl)pseudouridine TP —U NO 1-(4-Bromobenzyl)pseudouridine TP — U NO1-(4-Chlorobenzyl)pseudouridine TP — U NO1-(4-Fluorobenzyl)pseudouridine TP — U NO 1-(4-Iodobenzyl)pseudouridineTP — U NO 1-(4- — U NO Methanesulfonylbenzyl)pseudouridine TP1-(4-Methoxybenzyl)pseudouridine TP — U NO1-(4-Methoxy-benzyl)pseudo-UTP — U NO 1-(4-Methoxy-phenyl)pseudo-UTP — UNO 1-(4-Methylbenzyl)pseudouridine TP — U NO1-(4-Methyl-benzyl)pseudo-UTP — U NO 1-(4-Nitrobenzyl)pseudouridine TP —U NO 1-(4-Nitro-benzyl)pseudo-UTP — U NO 1(4-Nitro-phenyl)pseudo-UTP — UNO 1-(4- — U NO Thiomethoxybenzyl)pseudouridine TP 1-(4- — U NOTrifluoromethoxybenzyl)pseudouridine TP 1-(4- — U NOTrifluoromethylbenzyl)pseudouridine TP 1-(5-Amino-pentyl)pseudo-UTP — UNO 1-(6-Amino-hexyl)pseudo-UTP — U NO 1,6-Dimethyl-pseudo-UTP — U NO1-[3-(2-{2-[2-(2-Aminoethoxy)- — U NO ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP 1-{3-[2-(2-Aminoethoxy)-ethoxy]- — U NOpropionyl} pseudouridine TP 1-Acetylpseudouridine TP — U NO1-Alkyl-6-(1-propynyl)-pseudo-UTP — U NOl-Alkyl-6-(2-propynyl)-pseudo-UTP — U NO 1-Alkyl-6-allyl-pseudo-UTP — UNO 1-Alkyl-6-ethynyl-pseudo-UTP — U NO 1-Alkyl-6-homoallyl-pseudo-UTP —U NO 1-Alkyl-6-vinyl-pseudo-UTP — U NO 1-Allylpseudouridine TP — U NO1-Aminomethyl-pseudo-UTP — U NO 1-Benzoylpseudouridine TP — U NO1-Benzyloxymethylpseudouridine TP — U NO 1-Benzyl-pseudo-UTP — U NOl-Biotinyl-PEG2-pseudouridine TP — U NO 1-Biotinylpseudouridine TP — UNO 1-Butyl-pseudo-UTP — U NO 1-Cyanomethylpseudouridine TP — U NO1-Cyclobutylmethyl-pseudo-UTP — U NO 1-Cyclobutyl-pseudo-UTP — U NO1-Cycloheptylmethyl-pseudo-UTP — U NO 1-Cycloheptyl-pseudo-UTP — U NO1-Cyclohexylmethyl-pseudo-UTP — U NO 1-Cyclohexyl-pseudo-UTP — U NO1-Cyclooctylmethyl-pseudo-UTP — U NO 1-Cyclooctyl-pseudo-UTP — U NO1-Cyclopentylmethyl-pseudo-UTP — U NO 1-Cyclopentyl-pseudo-UTP — U NO1-Cyclopropylmethyl-pseudo-UTP — U NO 1-Cyclopropyl-pseudo-UTP — U NO1-Ethyl-pseudo-UTP — U NO 1-Hexyl-pseudo-UTP — U NO1-Homoallylpseudouridine TP — U NO 1-Hydroxymethylpseudouridine TP — UNO 1-iso-propyl-pseudo-UTP — U NO 1-Me-2-thio-pseudo-UTP — U NO1-Me-4-thio-pseudo-UTP — U NO 1-Me-alpha-thio-pseudo-UTP — U NO 1- — UNO Methanesulfonylmethylpseudouridine TP 1-Methoxymethylpseudouridine TP— U NO 1-Methyl-6-(2,2,2- — U NO Trifluoroethyl)pseudo-UTP1-Methyl-6-(4-morpholino)-pseudo- — U NO UTP1-Methyl-6-(4-thiomorpholino)- — U NO pseudo-UTP 1-Methyl-6-(substituted— U NO phenyl)pseudo-UTP 1-Methyl-6-amino-pseudo-UTP — U NO1-Methyl-6-azido-pseudo-UTP — U NO 1-Methyl-6-bromo-pseudo-UTP — U NO1-Methyl-6-butyl-pseudo-UTP — U NO 1-Methyl-6-chloro-pseudo-UTP — U NO1-Methyl-6-cyano-pseudo-UTP — U NO 1-Methyl-6-dimethylamino-pseudo- — UNO UTP 1-Methyl-6-ethoxy-pseudo-UTP — U NO1-Methyl-6-ethylcarboxylate-pseudo- — U NO UTP1-Methyl-6-ethyl-pseudo-UTP — U NO 1-Methyl-6-fluoro-pseudo-UTP — U NO1-Methyl-6-formyl-pseudo-UTP — U NO 1-Methyl-6-hydroxyamino-pseudo- — UNO UTP 1-Methyl-6-hydroxy-pseudo-UTP — U NO 1-Methyl-6-iodo-pseudo-UTP —U NO 1-Methyl-6-iso-propyl-pseudo-UTP — U NO1-Methyl-6-methoxy-pseudo-UTP — U NO 1-Methyl-6-methylamino-pseudo-UTP —U NO 1-Methyl-6-phenyl-pseudo-UTP — U NO 1-Methyl-6-propyl-pseudo-UTP —U NO 1-Methyl-6-tert-butyl-pseudo-UTP — U NO1-Methyl-6-trifluoromethoxy-pseudo- — U NO UTP1-Methyl-6-trifluoromethyl-pseudo- — U NO UTP1-Morpholinomethylpseudouridine TP — U NO 1-Pentyl-pseudo-UTP — U NO1-Phenyl-pseudo-UTP — U NO 1-Pivaloylpseudouridine TP — U NO1-Propargylpseudouridine TP — U NO 1-Propyl-pseudo-UTP — U NO1-propynyl-pseudouridine — U NO 1-p-tolyl-pseudo-UTP — U NO1-tert-Butyl-pseudo-UTP — U NO 1-Thiomethoxymethylpseudouridine — U NOTP 1- — U NO Thiomorpholinomethylpseudouridine TP1-Trifluoroacetylpseudouridine TP — U NO 1-Trifluoromethyl-pseudo-UTP —U NO 1-Vinylpseudouridine TP — U NO 2,2′-anhydro-uridine TP — U NO2′-bromo-deoxyuridine TP — U NO 2′-F-5-Methyl-2′-deoxy-UTP — U NO2′-OMe-5-Me-UTP — U NO 2′-OMe-pseudo-UTP — U NO 2′-a-Ethynyluridine TP —U NO 2′-a-Trifluoromethyluridine TP — U NO 2′-b-Ethynyluridine TP — U NO2′-b-Trifluoromethyluridine TP — U NO 2′-Deoxy-2′,2′-difluorouridine TP— U NO 2′-Deoxy-2′-a-mercaptouridine TP — U NO2′-Deoxy-2′-a-thiomethoxyuridine TP — U NO 2′-Deoxy-2′-b-aminouridine TP— U NO 2′-Deoxy-2′-b-azidouridine TP — U NO 2′-Deoxy-2′-b-bromouridineTP — U NO 2′-Deoxy-2′-b-chlorouridine TP — U NO2′-Deoxy-2′-b-fluorouridine TP — U NO 2′-Deoxy-2′-b-iodouridine TP — UNO 2′-Deoxy-2′-b-mercaptouridine TP — U NO2′-Deoxy-2′-b-thiomethoxyuridine TP — U NO 2-methoxy-4-thio-uridine — UNO 2-methoxyuridine — U NO 2′-O-Methyl-5-(1-propynyl)uridine TP — U NO3-Alkyl-pseudo-UTP — U NO 4′-Azidouridine TP — U NO 4′-Carbocyclicuridine TP — U NO 4′-Ethynyluridine TP — U NO 5-(1-Propynyl)ara-uridineTP — U NO 5-(2-Furanyl)uridine TP — U NO 5-Cyanouridine TP — U NO5-Dimethylaminouridine TP — U NO 5′-Homo-uridine TP — U NO5-iodo-2′-fluoro-deoxyuridine TP — U NO 5-Phenylethynyluridine TP — U NO5-Trideuteromethyl-6-deuterouridine — U NO TP 5-Trifluoromethyl-UridineTP — U NO 5-Vinylarauridine TP — U NO6-(2,2,2-Trifluoroethyl)-pseudo-UTP — U NO 6-(4-Morphoiino)-pseudo-UTP —U NO 6-(4-Thiomorpholino)-pseudo-UTP — U NO6-(Substituted-Phenyl)-pseudo-UTP — U NO 6-Amino-pseudo-UTP — U NO6-Azido-pseudo-UTP — U NO 6-Bromo-pseudo-UTP — U NO 6-Butyl-pseudo-UTP —U NO 6-Chloro-pseudo-UTP — U NO 6-Cyano-pseudo-UTP — U NO6-Dimethylamino-pseudo-UTP — U NO 6-Ethoxy-pseudo-UTP — U NO6-Ethylcarboxylate-pseudo-UTP — U NO 6-Ethyl-pseudo-UTP — U NO6-Fluoro-pseudo-UTP — U NO 6-Formyl-pseudo-UTP — U NO6-Hydroxyamino-pseudo-UTP — U NO 6-Hydroxy-pseudo-UTP — U NO6-Iodo-pseudo-UTP — U NO 6-iso-Propyl-pseudo-UTP — U NO6-Methoxy-pseudo-UTP — U NO 6-Methylamino-pseudo-UTP — U NO6-Methyl-pseudo-UTP — U NO 6-Phenyl-pseudo-UTP — U NO6-Phenyl-pseudo-UTP — U NO 6-Propyl-pseudo-UTP — U NO6-tert-Butyl-pseudo-UTP — U NO 6-Trifluoromethoxy-pseudo-UTP — U NO6-Trifluoromethyl-pseudo-UTP — U NO Alpha-thio-pseudo-UTP — U NOPseudouridine 1-(4- — U NO methylbenzenesulfonic acid) TP Pseudouridine1-(4-methylbenzoic — U NO acid) TP Pseudouridine TP 1-[3-(2- — U NOethoxy)]propionic acid Pseudouridine TP 1-[3-{2-(2-[2-(2- — U NOethoxy)-ethoxy]-ethoxy)- ethoxy}]propionic acid Pseudouridine TP1-[3-{2-(2-[2-{2(2- — U NO ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]- — U NOethoxy)-ethoxy}]propionic acid Pseudouridine TP 1-[3-{2-(2-ethoxy)- — UNO ethoxy}] propionic acid Pseudouridine TP 1-methylphosphonic — U NOacid Pseudouridine TP 1-methylphosphonic — U NO acid diethyl esterPseudo-UTP-N1-3-propionic acid — U NO Pseudo-UTP-N1-4-butanoic acid — UNO Pseudo-UTP-N1-5-pentanoic acid — U NO Pseudo-UTP-N1-6-hexanoic acid —U NO Pseudo-UTP-N1-7-heptanoic acid — U NOPseudo-UTP-N1-methyl-p-benzoic — U NO acid Pseudo-UTP-N1-p-benzoic acid— U NO Wybutosine yW W YES Hydroxywybutosine OHyW W YES Isowyosine imG2W YES Peroxywybutosine o2yW W YES undermodified hydroxywybutosine OHyW*W YES 4-demethylwyosine imG-14 W YES

In some embodiments, the modified nucleobase is pseudouridine (ψ),N1-methylpseudouridine (m¹ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine. In some embodiments, an RNA ofthe invention includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine(m¹ψ), 5-methoxy-uridine (m⁵U), 5-methyl-cytidine (m⁵C), pseudouridine(w), α-thio-guanosine, or α-thio-adenosine. In some embodiments, an mRNAof the invention includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In some embodiments, the RNA comprises pseudouridine (ψ) and5-methyl-cytidine (m⁵C). In some embodiments, the RNA comprises1-methyl-pseudouridine (m¹ψ). In some embodiments, the RNA comprises1-methyl-pseudouridine (m¹ψ) and 5-methyl-cytidine (m⁵C). In someembodiments, the RNA comprises 2-thiouridine (s²U). In some embodiments,the RNA comprises 2-thiouridine and 5-methyl-cytidine (m⁵C). In someembodiments, the RNA comprises 5-methoxy-uridine (mo⁵U). In someembodiments, the RNA comprises 5-methoxy-uridine (mo⁵U) and5-methyl-cytidine (m⁵C). In some embodiments, the RNA comprises2′-O-methyl uridine. In some embodiments, the RNA comprises 2′-O-methyluridine and 5-methyl-cytidine (m⁵C). In some embodiments, the RNAcomprises N6-methyl-adenosine (m⁶A). In some embodiments, the RNAcomprises N6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In certain embodiments, an RNA of the invention is uniformly modified(i.e., fully modified, modified throughout the entire sequence) for aparticular modification. For example, an RNA can be uniformly modifiedwith 5-methyl-cytidine (m⁵C), meaning that all cytosine residues in theRNA sequence are replaced with 5-methyl-cytidine (m⁵C). Similarly, RNAsof the invention can be uniformly modified for any type of nucleotideresidue present in the sequence by replacement with a modified residuesuch as those set forth above.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases, nucleosides, and nucleotides having a modifiedcytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C),5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine(hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine.

In some embodiments, the modified nucleobase is a modified uridine.Exemplary nucleobases, nucleosides, and nucleotides having a modifieduridine include 5-cyano uridine or 4′-thio uridine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases, nucleosides, and nucleotides having a modifiedadenine include 7-deaza-adenine, 1-methyl-adenosine (m1A),2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and2,6-Diaminopurine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases, nucleosides, and nucleotides having a modifiedguanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG),methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine(preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine(m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine,7-methyl-8-oxo-guanosine.

In one embodiment, the polynucleotides of the present invention, such asIVT polynucleotides, may have a uniform chemical modification of all orany of the same nucleotide type or a population of modificationsproduced by mere downward titration of the same starting modification inall or any of the same nucleotide type, or a measured percent of achemical modification of any of the same nucleotide type but with randomincorporation, such as where all uridines are replaced by a uridineanalog, e.g., pseudouridine. In another embodiment, the polynucleotidesmay have a uniform chemical modification of two, three, or four of thesame nucleotide type throughout the entire polynucleotide (such as alluridines and all cytosines, etc. are modified in the same way). When thepolynucleotides of the present invention are chemically and/orstructurally modified the polynucleotides may be referred to as“modified polynucleotides.”

Generally, the length of the IVT polynucleotide (e.g., IVT RNA) encodinga polypeptide of interest is greater than about 30 nucleotides in length(e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80,90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700,1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,80,000, 90,000 or up to and including 100,000 nucleotides).

In some embodiments, the IVT polynucleotide (e.g., IVT RNA) includesfrom about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000,from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000,from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000,from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000,from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to100,000).

In some embodiments, a nucleic acid as described herein is a chimericpolynucleotide. Chimeric polynucleotides or RNA constructs maintain amodular organization similar to IVT polynucleotides, but the chimericpolynucleotides comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thepolynucleotide. As such, the chimeric polynucleotides which are modifiedRNA molecules of the present invention are termed “chimeric modifiedRNA” or “chimeric RNA.” Chimeric polynucleotides have portions orregions which differ in size and/or chemical modification pattern,chemical modification position, chemical modification percent orchemical modification population and combinations of the foregoing.

Polypeptides of Interest

In some embodiments of the invention the is one or more of thefollowing: mRNA, modified mRNA, unmodified RNA, IncRNA, self-replicatingRNA, circular RNA, CRISPR guide RNA, and the like. In embodiments theRNA is RNA that encodes a polypeptide, such as mRNA or self-replicatingRNA.

In exemplary aspects of the invention, highly pure RNAs compositions areused to produce polypeptides of interest, e.g., therapeutic proteins,vaccine antigen, and the like. In some embodiments, the nucleic acidsare therapeutic RNAs. As used herein, the term “therapeutic mRNA” refersto an mRNA that encodes a therapeutic protein. Therapeutic proteinsmediate a variety of effects in a host cell or a subject in order totreat a disease or ameliorate the signs and symptoms of a disease. Forexample, a therapeutic protein can replace a protein that is deficientor abnormal, augment the function of an endogenous protein, provide anovel function to a cell (e.g., inhibit or activate an endogenouscellular activity, or act as a delivery agent for another therapeuticcompound (e.g., an antibody-drug conjugate). Therapeutic mRNA may beuseful for the treatment of the following diseases and conditions:bacterial infections, viral infections, parasitic infections, cellproliferation disorders, genetic disorders, and autoimmune disorders.

Thus, the polynucleotides of the invention can be used as therapeutic orprophylactic agents. They are provided for use in medicine. For example,the RNA described herein can be administered to a subject, wherein thepolynucleotides are translated in vivo to produce a therapeutic peptide.Provided are compositions, methods, kits, and reagents for diagnosis,treatment or prevention of a disease or condition in humans and othermammals. The active therapeutic agents of the invention include thepolynucleotides, cells containing polynucleotides or polypeptidestranslated from the polynucleotides.

The polynucleotides may be induced for translation in a cell, tissue ororganism. Such translation can be in vivo, ex vivo, in culture, or invitro. The cell, tissue or organism is contacted with an effectiveamount of a composition containing a polynucleotide which contains theRNA polynucleotides.

An “effective amount” of the polynucleotides are provided based, atleast in part, on the target tissue, target cell type, means ofadministration, physical characteristics of the polynucleotide (e.g.,size, and extent of modified nucleotides) and other components of thepolynucleotides, and other determinants. In general, an effective amountof the polynucleotides provides an induced or boosted peptide productionin the cell, preferably more efficient than a composition containing acorresponding unmodified polynucleotide encoding the same peptide.Increased peptide production may be demonstrated by increased celltransfection, increased protein translation from the polynucleotide,decreased nucleic acid degradation (as demonstrated, e.g., by increasedduration of protein translation from a modified polynucleotide), oraltered peptide production in the host cell.

The RNA of the present invention may be designed to encode polypeptidesof interest selected from any of several target categories including,but not limited to, biologics, antibodies, vaccines, therapeuticproteins or peptides, cell penetrating peptides, secreted proteins,plasma membrane proteins, cytoplasmic or cytoskeletal proteins,intracellular membrane bound proteins, nuclear proteins, proteinsassociated with human disease, targeting moieties or those proteinsencoded by the human genome for which no therapeutic indication has beenidentified but which nonetheless have utility in areas of research anddiscovery. “Therapeutic protein” refers to a protein that, whenadministered to a cell has a therapeutic, diagnostic, and/orprophylactic effect and/or elicits a desired biological and/orpharmacological effect.

The RNA disclosed herein, may encode one or more biologics. As usedherein, a “biologic” is a polypeptide-based molecule produced by themethods provided herein and which may be used to treat, cure, mitigate,prevent, or diagnose a serious or life-threatening disease or medicalcondition. Biologics, according to the present invention include, butare not limited to, allergenic extracts (e.g. for allergy shots andtests), blood components, gene therapy products, human tissue orcellular products used in transplantation, vaccines, monoclonalantibodies, cytokines, growth factors, enzymes, thrombolytics, andimmunomodulators, among others.

According to the present invention, one or more biologics currentlybeing marketed or in development may be encoded by the RNA of thepresent invention. While not wishing to be bound by theory, it isbelieved that incorporation of the encoding polynucleotides of a knownbiologic into the RNA of the invention will result in improvedtherapeutic efficacy due at least in part to the specificity, purityand/or selectivity of the construct designs.

The RNA disclosed herein, may encode one or more antibodies or fragmentsthereof. The term “antibody” includes monoclonal antibodies (includingfull length antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, diabodies, and single-chain molecules), aswell as antibody fragments. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein. As used herein, the term“monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations and/or post-translation modifications (e.g.,isomerizations, amidations) that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity. Chimericantibodies of interest herein include, but are not limited to,“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g., Old World Monkey, Apeetc.) and human constant region sequences.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies; nanobodies; single-chainantibody molecules and multispecific antibodies formed from antibodyfragments.

Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM,may be encoded by the RNA of the invention, including the heavy chainsdesignated alpha, delta, epsilon, gamma and mu, respectively. Alsoincluded are polynucleotide sequences encoding the subclasses, gamma andmu. Hence any of the subclasses of antibodies may be encoded in part orin whole and include the following subclasses: IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2. According to the present invention, one or moreantibodies or fragments currently being marketed or in development maybe encoded by the RNA of the present invention.

Antibodies encoded in the RNA of the invention may be utilized to treatconditions or diseases in many therapeutic areas such as, but notlimited to, blood, cardiovascular, CNS, poisoning (includingantivenoms), dermatology, endocrinology, gastrointestinal, medicalimaging, musculoskeletal, oncology, immunology, respiratory, sensory andanti-infective.

In one embodiment, RNA disclosed herein may encode monoclonal antibodiesand/or variants thereof. Variants of antibodies may also include, butare not limited to, substitutional variants, conservative amino acidsubstitution, insertional variants, deletional variants and/or covalentderivatives. In one embodiment, the RNA disclosed herein may encode animmunoglobulin Fc region. In another embodiment, the RNA may encode avariant immunoglobulin Fc region.

ThemRNA disclosed herein, may encode one or more vaccine antigens. Asused herein, a “vaccine antigen” is a biological preparation thatimproves immunity to a particular disease or infectious agent. Accordingto the present invention, one or more vaccine antigens currently beingmarketed or in development may be encoded by the RNA of the presentinvention.

Vaccine antigens encoded in the RNA of the invention may be utilized totreat conditions or diseases in many therapeutic areas such as, but notlimited to, cancer, allergy and infectious disease. In some embodimentsthe cancer vaccines may be personalized cancer vaccines in the form of aconcatemer or individual RNAs encoding peptide epitopes or a combinationthereof.

The RNA of the present invention may be designed to encode on or moreantimicrobial peptides (AMP) or antiviral peptides (AVP). AMPs and AVPshave been isolated and described from a wide range of animals such as,but not limited to, microorganisms, invertebrates, plants, amphibians,birds, fish, and mammals. The anti-microbial polypeptides describedherein may block cell fusion and/or viral entry by one or more envelopedviruses (e.g., HIV, HCV). For example, the anti-microbial polypeptidecan comprise or consist of a synthetic peptide corresponding to aregion, e.g., a consecutive sequence of at least about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the transmembranesubunit of a viral envelope protein, e.g., HIV-1 gp120 or gp41. Theamino acid and nucleotide sequences of HIV-1 gp120 or gp41 are describedin, e.g., Kuiken et al., (2008). “HIV Sequence Compendium,” Los AlamosNational Laboratory.

In some embodiments, the anti-microbial polypeptide may have at leastabout 75%, 80%, 85%, 9%, 95%, 100% sequence homology to thecorresponding viral protein sequence. In some embodiments, theanti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%,95%, or 100% sequence homology to the corresponding viral proteinsequence.

In other embodiments, the anti-microbial polypeptide may comprise orconsist of a synthetic peptide corresponding to a region, e.g., aconsecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 amino acids of the binding domain of a capsid bindingprotein. In some embodiments, the anti-microbial polypeptide may have atleast about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to thecorresponding sequence of the capsid binding protein.

The anti-microbial polypeptides described herein may block proteasedimerization and inhibit cleavage of viral proproteins (e.g., HIVGag-pol processing) into functional proteins thereby preventing releaseof one or more enveloped viruses (e.g., HIV, HCV). In some embodiments,the anti-microbial polypeptide may have at least about 75%, 800/%, 85%,90%, 95%, 100% sequence homology to the corresponding viral proteinsequence.

In other embodiments, the anti-microbial polypeptide can comprise orconsist of a synthetic peptide corresponding to a region, e.g., aconsecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 amino acids of the binding domain of a proteasebinding protein. In some embodiments, the anti-microbial polypeptide mayhave at least about 75%, 80%0, 85%, 90%, 95%, 100% sequence homology tothe corresponding sequence of the protease binding protein.

A non-limiting list of infectious diseases that the RNA vaccine antigensor anti-microbial peptides may treat is presented below: humanimmunodeficiency virus (HIV), HIV resulting in mycobacterial infection,AIDS related Cacheixa, AIDS related Cytomegalovirus infection,HIV-associated nephropathy, Lipodystrophy, AID related cryptococcalmeningitis, AIDS related neutropaenia, Pneumocysitisjiroveci(Pneumocystis carinii) infections, AID related toxoplasmosis, hepatitisA, B, C, D or E, herpes, herpes zoster (chicken pox), German measles(rubella virus), yellow fever, dengue fever etc. (flavi viruses), flu(influenza viruses), haemorrhagic infectious diseases (Marburg or Ebolaviruses), bacterial infectious diseases such as Legionnaires' disease(Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), E. coliinfections, staphylococcal infections, salmonella infections orstreptococcal infections, tetanus (Clostridium tetani), protozoaninfectious diseases (malaria, sleeping sickness, leishmaniasis,toxoplasmosis, i.e. infections caused by plasmodium, trypanosomes,leishmania and toxoplasma), diphtheria, leprosy, measles, pertussis,rabies, tetanus, tuberculosis, typhoid, varicella, diarrheal infectionssuch as Amoebiasis, Clostridium difficile-associated diarrhea (CDAD),Cryptosporidiosis, Giardiasis, Cyclosporiasis and Rotaviralgastroenteritis, encephalitis such as Japanese encephalitis, Westerequine encephalitis and Tick-borne encephalitis (TBE), fungal skindiseases such as candidiasis, onychomycosis, Tinea captis/scal ringworm,Tinea corporis/body ringworm, Tinea cruris/jock itch, sporotrichosis andTinea pedis/Athlete's foot, Meningitis such as Haemophilus influenzatype b (Hib), Meningitis, viral, meningococcal infections andpneumococcal infection, neglected tropical diseases such as Argentinehaemorrhagic fever, Leishmaniasis, Nematode/roundworm infections, Rossriver virus infection and West Nile virus (WNV) disease, Non-HIV STDssuch as Trichomoniasis, Human papillomavirus (HPV) infections, sexuallytransmitted chlamydial diseases, Chancroid and Syphilis, Non-septicbacterial infections such as cellulitis, lyme disease, MRSA infection,pseudomonas, staphylococcal infections, Boutonneuse fever,Leptospirosis, Rheumatic fever, Botulism, Rickettsial disease andMastoiditis, parasitic infections such as Cysticercosis, Echinococcosis,Trematode/Fluke infections, Trichinellosis, Babesiosis, Hypodermyiasis,Diphyllobothriasis and Trypanosomiasis, respiratory infections such asadenovirus infection, aspergillosis infections, avian (H5N1) influenza,influenza, RSV infections, severe acute respiratory syndrome (SARS),sinusitis, Legionellosis, Coccidioidomycosis and swine (H1N1) influenza,sepsis such as bacteraemia, sepsis/septic shock, sepsis in prematureinfants, urinary tract infection such as vaginal infections (bacterial),vaginal infections (fungal) and gonococcal infection, viral skindiseases such as B19 parvovirus infections, warts, genital herpes,orofacial herpes, shingles, inner ear infections, fetal cytomegalovirussyndrome, foodborn illnesses such as brucellosis (Brucella species),Clostridium perfringens (Epsilon toxin), E. Coli O157:H7 (Escherichiacoli), Salmonellosis (Salmonella species), Shingellosis (Shingella),Vibriosis and Listeriosis, bioterrorism and potential epidemic diseasessuch as Ebola haemorrhagic fever, Lassa fever, Marburg haemorrhagicfever, plague, Anthrax Nipah virus disease, Hanta virus, Smallpox,Glanders (Burkholderia mallei), Melioidosis (Burkholderia pseudomallei),Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), Tularemia(Fancisella tularensis), rubella, mumps and polio.

The RNA disclosed herein, may encode one or more validated or “intesting” therapeutic proteins or peptides. According to the presentinvention, one or more therapeutic proteins or peptides currently beingmarketed or in development may be encoded by the RNA of the presentinvention. Therapeutic proteins and peptides encoded in the RNA of theinvention may be utilized to treat conditions or diseases in manytherapeutic areas such as, but not limited to, blood, cardiovascular,CNS, poisoning (including antivenoms), dermatology, endocrinology,genetic, genitourinary, gastrointestinal, musculoskeletal, oncology, andimmunology, respiratory, sensory and anti-infective.

The RNA disclosed herein, may encode one or more cell-penetratingpolypeptides. As used herein, “cell-penetrating polypeptide” or CPPrefers to a polypeptide which may facilitate the cellular uptake ofmolecules. A cell-penetrating polypeptide of the present invention maycontain one or more detectable labels. The polypeptides may be partiallylabeled or completely labeled throughout. The RNA may encode thedetectable label completely, partially or not at all. Thecell-penetrating peptide may also include a signal sequence. As usedherein, a “signal sequence” refers to a sequence of amino acid residuesbound at the amino terminus of a nascent protein during proteintranslation. The signal sequence may be used to signal the secretion ofthe cell-penetrating polypeptide.

In one embodiment, the RNA may also encode a fusion protein. The fusionprotein may be created by operably linking a charged protein to atherapeutic protein. As used herein, “operably linked” refers to thetherapeutic protein and the charged protein being connected in such away to permit the expression of the complex when introduced into thecell. As used herein, “charged protein” refers to a protein that carriesa positive, negative or overall neutral electrical charge. Preferably,the therapeutic protein may be covalently linked to the charged proteinin the formation of the fusion protein. The ratio of surface charge tototal or surface amino acids may be approximately 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8 or 0.9.

The cell-penetrating polypeptide encoded by the RNA may form a complexafter being translated. The complex may comprise a charged proteinlinked, e.g. covalently linked, to the cell-penetrating polypeptide.

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but is not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the RNA may be introduced. The cell-penetrating polypeptidemay also be capable of penetrating the first cell.

In one embodiment, the RNA may encode a cell-penetrating polypeptidewhich may comprise a protein-binding partner. The protein bindingpartner may include, but is not limited to, an antibody, a superchargedantibody or a functional fragment. The RNA may be introduced into thecell where a cell-penetrating polypeptide comprising the protein-bindingpartner is introduced.

Human and other eukaryotic cells are subdivided by membranes into manyfunctionally distinct compartments. Each membrane-bound compartment, ororganelle, contains different proteins essential for the function of theorganelle. The cell uses “sorting signals” which are amino acid motifslocated within the protein, to target proteins to particular cellularorganelles. One type of sorting signal, called a signal sequence, asignal peptide, or a leader sequence, directs a class of proteins to anorganelle called the endoplasmic reticulum (ER).

Proteins targeted to the ER by a signal sequence can be released intothe extracellular space as a secreted protein. Similarly, proteinsresiding on the cell membrane can also be secreted into theextracellular space by proteolytic cleavage of a “linker” holding theprotein to the membrane. While not wishing to be bound by theory, themolecules of the present invention may be used to exploit the cellulartrafficking described above. As such, in some embodiments of theinvention, RNA are provided to express a secreted protein. In oneembodiment, these may be used in the manufacture of large quantities ofvaluable human gene products.

In some embodiments of the invention, RNA are provided to express aprotein of the plasma membrane.

In some embodiments of the invention, RNA are provided to express acytoplasmic or cytoskeletal protein.

In some embodiments of the invention, RNA are provided to express anintracellular membrane bound protein.

In some embodiments of the invention, RNA are provided to express anuclear protein.

In some embodiments of the invention, RNA are provided to express aprotein associated with human disease.

The RNA may have a nucleotide sequence of a native or naturallyoccurring RNA or encoding a native or naturally occurring peptide.Alternatively, the RNA may have a nucleotide sequence having a percentidentity to the nucleotide sequence of a native or naturally occurringRNA or mRNA may have a nucleotide sequence encoding a peptide having apercent identity to the nucleotide sequence of a native or naturallyoccurring peptide. The term “identity” as known in the art, refers to arelationship between the sequences of two or more peptides, asdetermined by comparing the sequences. In the art, identity also meansthe degree of sequence relatedness between peptides, as determined bythe number of matches between strings of two or more amino acidresidues. Identity measures the percent of identical matches between thesmaller of two or more sequences with gap alignments (if any) addressedby a particular mathematical model or computer program (i.e.,“algorithms”). Identity of related peptides can be readily calculated byknown methods. Such methods include, but are not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991;and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

Thus, in some embodiments, the peptides encoded by the RNAs arepolypeptide variants that may have the same or a similar activity as areference polypeptide. Alternatively, the variant may have an alteredactivity (e.g., increased or decreased) relative to a referencepolypeptide. Generally, variants of a particular polynucleotide orpolypeptide of the invention will have at least about 4%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% but less than 100% sequence identity to that particularreference polynucleotide or polypeptide as determined by sequencealignment programs and parameters described herein and known to thoseskilled in the art. Such tools for alignment include those of the BLASTsuite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer,Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997),“Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25:3389-3402.) Other tools are describedherein, specifically in the definition of “Identity.” Default parametersin the BLAST algorithm include, for example, an expect threshold of 10,Word size of 28, Match/Mismatch Scores 1, -2, Gap costs Linear. Anyfilter can be applied as well as a selection for species specificrepeats, e.g., Homo sapiens.

According to the present invention, the polynucleotides include RNA toencode one or more polypeptides of interest or fragments thereof. Apolypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides.As used herein, the term “polypeptides of interest” refer to anypolypeptide which is selected to be encoded in the primary construct ofthe present invention. As used herein, “polypeptide” means a polymer ofamino acid residues (natural or unnatural) linked together most often bypeptide bonds. The term, as used herein, refers to proteins,polypeptides, and peptides of any size, structure, or function. In someinstances, the polypeptide encoded is smaller than about 50 amino acidsand the polypeptide is then termed a peptide. If the polypeptide is apeptide, it will be at least about 2, 3, 4, or at least 5 amino acidresidues long. Thus, polypeptides include gene products, naturallyoccurring polypeptides, synthetic polypeptides, homologs, orthologs,paralogs, fragments and other equivalents, variants, and analogs of theforegoing. A polypeptide may be a single molecule or may be amulti-molecular complex such as a dimer, trimer or tetramer. They mayalso comprise single chain or multichain polypeptides such as antibodiesor insulin and may be associated or linked. Most commonly disulfidelinkages are found in multichain polypeptides. The term polypeptide mayalso apply to amino acid polymers in which one or more amino acidresidues are an artificial chemical analogue of a correspondingnaturally occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity to a native or reference sequence,and preferably, they will be at least about 80%, more preferably atleast about 90% identical to a native or reference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, RNA encoding polypeptides containing substitutions, insertionsand/or additions, deletions and covalent modifications with respect toreference sequences, in particular the polypeptide sequences disclosedherein, are included within the scope of this invention. For example,sequence tags or amino acids, such as one or more lysines, can be addedto the peptide sequences of the invention (e.g., at the N-terminal orC-terminal ends). Sequence tags can be used for peptide purification orlocalization. Lysines can be used to increase peptide solubility or toallow for biotinylation. Alternatively, amino acid residues located atthe carboxy and amino terminal regions of the amino acid sequence of apeptide or protein may optionally be deleted providing for truncatedsequences. Certain amino acids (e.g., C-terminal or N-terminal residues)may alternatively be deleted depending on the use of the sequence, asfor example, expression of the sequence as part of a larger sequencewhich is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the RNA of the invention,any of several manipulations and/or modifications of these features maybe performed by moving, swapping, inverting, deleting, randomizing orduplicating. Furthermore, it is understood that manipulation of featuresmay result in the same outcome as a modification to the molecules of theinvention. For example, a manipulation which involved deleting a domainwould result in the alteration of the length of a molecule just asmodification of a nucleic acid to encode less than a full lengthmolecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

Formulations/Pharmaceutical Compositions

The present invention provides polynucleotides and pharmaceuticalcompositions thereof optionally in combination with one or morepharmaceutically acceptable excipients. Pharmaceutical compositions mayoptionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances.Pharmaceutical compositions of the present invention may be sterileand/or pyrogen-free. General considerations in the formulation and/ormanufacture of pharmaceutical agents may be found, for example, inRemington: The Science and Practice of Pharmacy 21st ed., LippincottWilliams & Wilkins, 2005 (incorporated herein by reference in itsentirety).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to the polynucleotides,e.g., mRNA encoding polynucleotides to be delivered as described herein.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

The polynucleotides of the invention can be formulated using one or moreexcipients to: (1) increase stability; (2) increase cell transfection;(3) permit the sustained or delayed release (e.g., from a depotformulation); (4) alter the biodistribution (e.g., target to specifictissues or cell types); (5) increase the translation of encoded proteinin vivo; and/or (6) alter the release profile of encoded protein invivo. In addition to traditional excipients such as any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, excipients of thepresent invention can include, without limitation, lipidoids, liposomes,lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles,peptides, proteins, cells transfected with polynucleotides,hyaluronidase, nanoparticle mimics and combinations thereof.

In some embodiments, nucleic acid molecules of the invention can beformulated using one or more liposomes, lipoplexes, or lipidnanoparticles. In one embodiment, pharmaceutical compositions of nucleicacid molecules include lipid nanoparticles (LNPs). In some embodiments,lipid nanoparticles are MC3-based lipid nanoparticles.

In one embodiment, the polynucleotides may be formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished by methods known in the art. As a non-limitingexample, the polycation may include a cationic peptide or a polypeptidesuch as, but not limited to, polylysine, polyornithine and/orpolyarginine. In another embodiment, the polynucleotides may beformulated in a lipid-polycation complex which may further include anon-cationic lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation was composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety). In someembodiments, liposome formulations may comprise from about 35 to about45% cationic lipid, from about 40% to about 50% cationic lipid, fromabout 50°/% to about 60% cationic lipid and/or from about 55% to about65% cationic lipid. In some embodiments, the ratio of lipid to RNA inliposomes may be from about 5:1 to about 20:1, from about 10:1 to about25:1, from about 15:1 to about 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain from about 0.5% toabout 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0%and/or from about 3.0% to about 6.0% of the lipid molar ratio ofPEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In another embodiment the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the polynucleotide is formulated in a nanoparticlewhich may comprise at least one lipid. The lipid may be selected from,but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200,DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipidsand amino alcohol lipids. In another aspect, the lipid may be a cationiclipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA,DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationiclipid may be the lipids described in and/or made by the methodsdescribed in US Patent Publication No. US20130150625, hereinincorporated by reference in its entirety. As a non-limiting example,the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 inUS20130150625); or any pharmaceutically acceptable salt or stereoisomerthereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In one embodiment, the lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC. DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one embodiment, the formulation includes from about 25% to about 75%on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In one embodiment, the formulation includes from about 0.5% to about 15%on a molar basis of the neutral lipid e.g., from about 3 to about 12%,from about 5 to about 10% or about 15%, about 10%, or about 7.5% on amolar basis. Exemplary neutral lipids include, but are not limited to,DSPC, POPC, DPPC, DOPE and SM. In one embodiment, the formulationincludes from about 5% to about 50% on a molar basis of the sterol(e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol ischolesterol. In one embodiment, the formulation includes from about 0.5%to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g.,about 0.5 to about 10%, about 0.5 to about 5%/N, about 1.5%, about 0.5%,about 1.5%, about 3.5%, or about 5% on a molar basis. In one embodiment,the PEG or PEG modified lipid comprises a PEG molecule of an averagemolecular weight of 2,000 Da. In other embodiments, the PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof less than 2,000, for example around 1,500 Da, around 1,000 Da, oraround 500 Da. Exemplary PEG-modified lipids include, but are notlimited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein asPEG-C14 or C14-PEG), PEG-cDMA.

In one embodiment, the formulations of the inventions include 25-75% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include 35-65% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include 45-65% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about 60%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of the neutral lipid, about 31% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about500/% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 38.5% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about500/% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 35%/o of the sterol, about 4.5% or about 5% ofthe PEG or PEG-modified lipid, and about 0.5% of the targeting lipid ona molar basis.

In one embodiment, the formulations of the inventions include about 40%°of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% ofthe neutral lipid, about 40% of the sterol, and about 5% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about57.2% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1%of the neutral lipid, about 34.3% of the sterol, and about 1.4% of thePEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the inventions include about57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA(PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release,107, 276-287 (2005), the contents of which are herein incorporated byreference in its entirety), about 7.5% of the neutral lipid, about 31.5%of the sterol, and about 3.5% of the PEG or PEG-modified lipid on amolar basis.

In preferred embodiments, lipid nanoparticle formulation consistsessentially of a lipid mixture in molar ratios of about 20-70% cationiclipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modifiedlipid; more preferably in a molar ratio of about 20-60% cationiclipid:5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modifiedlipid.

In particular embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Exemplary lipid nanoparticle compositions and methods of making same aredescribed, for example, in Semple et al. (2010) Nat. Biotechnol.28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (thecontents of each of which are incorporated herein by reference in theirentirety).

In one embodiment, the lipid nanoparticle formulations described hereinmay comprise a cationic lipid, a PEG lipid and a structural lipid andoptionally comprise a non-cationic lipid. As a non-limiting example, thelipid nanoparticle may comprise about 40-60%0/of cationic lipid, about5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about30-50% of a structural lipid. As another non-limiting example, the lipidnanoparticle may comprise about 50% cationic lipid, about 10%non-cationic lipid, about 1.5% PEG lipid and about 38.5% structurallipid. As yet another non-limiting example, the lipid nanoparticle maycomprise about 55% cationic lipid, about 10% non-cationic lipid, about2.5% PEG lipid and about 32.5% structural lipid. In one embodiment, thecationic lipid may be any cationic lipid described herein such as, butnot limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In one embodiment, the lipid nanoparticle formulations described hereinmay be 4 component lipid nanoparticles. The lipid nanoparticle maycomprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise about 40-60% of cationic lipid, about 5-15% of a non-cationiclipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.As another non-limiting example, the lipid nanoparticle may compriseabout 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEGlipid and about 38.5% structural lipid. As yet another non-limitingexample, the lipid nanoparticle may comprise about 55% cationic lipid,about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5%structural lipid. In one embodiment, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In one embodiment, the lipid nanoparticle formulations described hereinmay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticlecomprise about 50% of the cationic lipid DLin-KC2-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about38.5% of the structural lipid cholesterol. As a non-limiting example,the lipid nanoparticle comprise about 50% of the cationic lipidDLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% ofthe PEG lipid PEG-DOMG and about 38.5% of the structural lipidcholesterol. As a non-limiting example, the lipid nanoparticle compriseabout 50% of the cationic lipid DLin-MC3-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about38.5% of the structural lipid cholesterol. As yet another non-limitingexample, the lipid nanoparticle comprise about 55% of the cationic lipidL319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEGlipid PEG-DMG and about 32.5% of the structural lipid cholesterol.

In one embodiment, the polynucleotides of the invention may beformulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In one embodiment, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm. In one embodiment, the lipid nanoparticle may have adiameter greater than 100 nm, greater than 150 nm, greater than 200 nm,greater than 250 nm, greater than 300 nm, greater than 350 nm, greaterthan 400 nm, greater than 450 nm, greater than 500 nm, greater than 550nm, greater than 600 nm, greater than 650 nm, greater than 700 nm,greater than 750 nm, greater than 800 nm, greater than 850 nm, greaterthan 900 nm, greater than 950 nm or greater than 1000 nm. In someembodiments, the cationic lipid nanoparticle has a mean diameter of50-150 nm. In some embodiments, the cationic lipid nanoparticle has amean diameter of 80-100 nm.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient. By way ofexample, the composition may comprise between 0.1% and 100%, e.g.,between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w)active ingredient.

In one embodiment, the compositions containing the polynucleotidesdescribed herein, formulated in a lipid nanoparticle comprising MC3,Cholesterol, DSPC and PEG2000-DMG, the buffer trisodium citrate, sucroseand water for injection. As a non-limiting example, the compositioncomprises: 2.0 mg/mL of drug substance, 21.8 mg/mL of MC3, 10.1 mg/mL ofcholesterol, 5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16 mg/mL oftrisodium citrate, 71 mg/mL of sucrose and about 1.0 mL of water forinjection.

The polynucleotides of the present invention may be administered by anyroute which results in a therapeutically effective outcome. The presentinvention provides methods comprising administering polynucleotides andin accordance with the invention to a subject in need thereof. The exactamount required will vary from subject to subject, depending on thespecies, age, and general condition of the subject, the severity of thedisease, the particular composition, its mode of administration, itsmode of activity, and the like. Compositions in accordance with theinvention are typically formulated in dosage unit form for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of the compositions of the present inventionmay be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective,prophylactically effective, or appropriate imaging dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging. The desired dosage may be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used.

A polynucleotide pharmaceutical composition described herein can beformulated into a dosage form described herein, such as an intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal,and subcutaneous).

The present invention provides pharmaceutical compositions includingpolynucleotides (e.g., RNA molecules) and polynucleotide compositionsand/or complexes optionally in combination with one or morepharmaceutically acceptable excipients.

The present invention provides polynucleotides (e.g., RNA molecules) andrelated pharmaceutical compositions and complexes optionally incombination with one or more pharmaceutically acceptable excipients.Pharmaceutical compositions may optionally comprise one or moreadditional active substances, e.g., therapeutically and/orprophylactically active substances. Pharmaceutical compositions of thepresent invention may be sterile and/or pyrogen-free. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporatedherein by reference in its entirety).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to the polynucleotides(e.g., RNA molecules), to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.,non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES Example 1. Manufacture of Polynucleotides

According to the present invention, the manufacture of polynucleotidesand or parts or regions thereof may be accomplished utilizing themethods taught in WO2014/152027 filed Mar. 15, 2013 entitled“Manufacturing Methods for Production of RNA Transcripts” (AttorneyDocket number M500), the contents of which is incorporated herein byreference in its entirety.

Purification methods may include those taught in InternationalApplication WO2014/152030 and WO2014/152031, each of which isincorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in WO2014/144039, which is incorporated herein byreference in its entirety.

Characterization of the polynucleotides of the disclosure may beaccomplished using a procedure selected from the group consisting ofpolynucleotide mapping, reverse transcriptase sequencing, chargedistribution analysis, and detection of RNA impurities, whereincharacterizing comprises determining the RNA transcript sequence,determining the purity of the RNA transcript, or determining the chargeheterogeneity of the RNA transcript. Such methods are taught in, forexample, WO2014/144711 and WO2014/144767, the contents of each of whichis incorporated herein by reference in its entirety.

Example 2. Chimeric Polynucleotide Synthesis Introduction

According to the present invention, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry.

According to this method, a first region or part of 100 nucleotides orless is chemically synthesized with a 5′ monophosphate and terminal3′desOH or blocked OH. If the region is longer than 80 nucleotides, itmay be synthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 130 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase,followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then it is preferable that such region or part comprise aphosphate-sugar backbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide is made using a series of starting segments.Such segments include:

(a) Capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) 5′ triphosphate segment which may include the coding region of apolypeptide and comprising a normal 3′OH (SEG. 2)

(c) 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) is treated withcordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) is then ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG.2-SEG. 3 construct is thenpurified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3: PCR for Production of DNA Template

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA −100 ng; and dH₂0 dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NANODROP™ andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 4. IVT and IFN-β Analysis of Short Model RNA-1

IVT reactions can be “spiked” with specific nucleotides, resulting inminimal cytokine response contaminants and better yields. In oneprocess, the GTP ratio to other NTPs was increased, resulting in ahigher total NPT load and an increased NTP:Mg²⁺ molar ratio, as shown inTable 1. These specific nucleotides are referred to as short model RNAs.The goal of short model RNAs was to recapitulate the same cytokinesignal/effect of equimolar (prior art) vs methods of the inventionobserved in full length RNAs. The construct was small enough to detectfull length impurities by LC/MS which was not currently feasible withfull length RNA.

TABLE 1 Nucleotide Formulations GDP Equimolar Alpha alpha [GDP] mM 0 030 [GTP] mM 7.5 30 15 [ATP] mM 7.5 15 15 [CTP] mM 7.5 7.5 7.5 [UTP] mM7.5 7.5 7.5 Total 30 60 75 [Nuc] mM [Mg2+] 40 40 40 mM Nuc: Mg2+ 0.751.50 1.88 Effective 90 180 195 [phosphate] T7 (U/μl 7 14 14 reaction)

In order to determine whether a short model transcript prepared usingtwo different IVT processes mimic the in vitro (BJ fibroblast) IFNbetaresponse that was observed for full length transcripts, independent ofexperimental process (i.e. crude IVT, ultrafiltered, or dT purified)study was set up. A surrogate RNA, short model RNA-1, produced under thesame IVT conditions as the target RNA and from the same DNA templateduplex, was generated. The cytokine response mimicked that of fulllength RNA in an IFN-β assay in BJ fibroblasts (FIG. 1). The short modeltranscript prepared using the equimolar IVT process has a higher IFNbetaresponse than material prepared using the alpha IVT process. Thecytokine response was preserved after ultrafiltration and dTpurification.

Furthermore, impurity profiling of short model RNA-1 (all uridinesmodified to pseudouridine and all cytidnes modified by 5′O-methyl),showed the presence of reverse complements (dsRNA) in the equimolarprocess but not in Alpha reaction. Nine different species wereidentified—the overlapping peaks between the two represent abortivespecies, while the peaks only seen in the equimolar formulation arereverse complementary species. The screen was performed on species withvarying amounts of uridine and cytidine modifications, and reversecomplementary and abortive species were found in all three (FIG. 2). Theabortive transcripts are sense and the reverse complementary areantisense transcripts. IFN-β levels also varied between the three (FIG.2). The top is G0=Standard A, G, C, U, Mid is G5=Standard A, G, C and1-methylpseudoU and Bottom is G6=Standard A, G, C and 5-methoxyuridine.Short model transcript prepared by 2 different processes in 3 differentchemistries have similar impurity profiles by LCMS, despite differencesin the BJF assay, which seems to be sensitive to different chemistries.

An LCMS analysis of a model transcript and intact transcript wereprepared at 25 C for 6 h IVT. A short transcript and full lengthtranscript were prepared using the equimolar and alpha processes at 25 Cfor 6 h (not our normal IVT, which is 37 C 2 h). Low temperature IVTreactions (<30 C) produce a larger abundance of reverse complement RNAsthan 37 C. The impurity profiles of the samples were analyzed by LCMS.Samples prepared using both processes both contained abortive/truncatedproducts. The transcripts prepared using equimolar process containedpolyU species of varying lengths. Alpha process still mitigates theformation of reverse complements even at 25 C where abundance wasgreater than standard conditions using equimolar processes (FIG. 3).

Detection of Complementary Antisense RNA

Immunostimulatory impurities appear to be driven by RNA-templated RNAtranscription, as T7 polymerizes off of the nascent transcribed RNA. Theresulting complementary/antisense RNA (dsRNA) that was generated showsmixed bases, a polyU component, and a 5′ triphosphate (5′ppp), whichinitiates with any base. An RNA-templated transcription was performed.Reverse phase purified hEPO G5 (cold) was incubated at 4 mg/mL(consistent with a typical yield of an in vitro transcription reaction),with all the IVT components, except DNA template, which was determinedto be below threshold required for in vitro transcription by qPCR, toexplore the RNA-templated transcription phenomenon. The materials wereanalyzed by UPLC and in vitro BJF IFNbeta assay. UPLC analysisdemonstrated that aberrant RNA transcription products, and most notablypolyU species are produced via RNA-templated transcription (in theabsence of DNA template). The reaction performed with RNA and all IVTcomponents was IFNbeta hot, while the reaction performed without RNA wascold. (FIG. 4).

Double-stranded RNA can be cleared from reverse-phase (RP) purifiedtranscribed RNA. A Capillary Electrophoresis analysis of RNase IIItreated hEPO G5 material was performed. hEPO G5 was prepared usingeither equimolar or alpha conditions and a portion was purified by RP.The samples were then treated with RNase III and analyzed by capillaryelectrophoresis. Material prepared by alpha process contained less RNaseIII substrate than that prepared by equimolar process. RP purificationclears most of the RNase III substrate from equi and alpha materialAlpha process and Reverse Phase purification appear to provide asynergistic reduction in RNase III substrate (eg dsRNA) (FIG. 5A). ACapillary Electrophoresis analysis of RNase III treated hEPO G0 materialwas performed. hEPO G0 was prepared using equimolar process and aportion was purified by RP. The samples were then treated with RNase IIIand analyzed by capillary electrophoresis (blue: treated; black:untreated). Material that was RP purified contained less RNase IIIsubstrate than material that was not RP purified (current state of theart) (FIG. 5B).

RNase III is a dsRNA specific nuclease. RNA preps are subjected to RNaseIII treatment for a fixed time. RNA Purity/impurity profile are comparedpre and post RNase III treatment and are measured by HPLC or capillaryelectrophoresis. The amount of full length product degraded wasproportional to the level of double stranded RNA impurities present inthe RNA prep. The amount degraded as indicated by a change in apparentsize/retention time/was considered to be amount of RNase III substrate.Samples devoid of dsRNA should show nearly identical purity pre and postRNase III treatment. Samples containing significant quantities of dsRNAwill show the formation of substantial cleavage product and depletion offull length RNA present in the untreated feedstock as seen by HPLC orcapillary electrophoresis. (Ex If 80% of the original mRNA remains postRNase III treatment, 20% was a substrate for RNase III.

An IFNbeta and hEPO expression analysis of samples from FIGS. 5A and 5Bwas performed. Treated and untreated samples were analyzed to determinehow RNase III treatment affect IFNbeta response and hEPO expression inBJ Fibroblasts (in vitro). It was found that hEPO A100 G5 equimolarmaterial expresses similarly for untreated and RIII treated samples.Cytokine level for equi treated −RP sample reduced after treatment withRIII. hEPO A100 G5 alpha material all expressed similarly and hadzero/low IFNbeta response. TL material does not express. However, RIIItreatment brought CK level down for both + and −RP samples. G0 hEPO A100material saw the greatest effects from RNase III treatment. After RNaseIII treatment, both + and −RP purified samples saw an increase inexpression and a decrease in IFNbeta level (FIG. 6).

A Capillary Electrophoresis analysis of a short transcript transcribedusing different processes and treated with RNase III was performed. Theinquiry was whether a short, model transcript may be used tocharacterize the effect of RNase III treatment? Surrogate RNA constructwas transcribed using equimolar or alpha processes then treated withRNase III and analyzed by capillary electrophoresis. Equimolar materialappeared to contain the most RNase III substrate, while alpha processmaterial did not contain any substrate, according to CE. With RNase IIItreatment, the model RNA peak shifts 5-7 nucleotides on the fragmentanalyzer. The equimolar IVT forms a drastic second peak 216 nucleotidesshorter than the main peak, which was also observed with the ORFcontaining mRNA (FIG. 7). Therefore, using model RNAs and LC/MS, one cancharacterize precisely what component was cut, and by deduction, whatcomponent remains. The HPLC purity method showed polyU upon isolationand enrichment of cleavage products (FIG. 9). An RP analysis of RNASurrogate 2 EQ transcript treated with RNase III (same construct as topfigure in FIG. 7 . . . alternative analysis) was performed. Severalspecies were observed by RP analysis after treatment of Surrogate RNAtranscript with RNase III. Since this RP method was a tail-selectivemethod, we hypothesize that these early eluting peaks are short oligosand/or tail variants (FIG. 8A). An RP analysis of RNA Surrogate 2 alphaconstruct transcribed using alpha process transcript treated with RNaseIII was performed. We did not observe any evolution of additionalimpurity peaks peaks and appreciable changes to the overall purity inthe RP trace for Surrogate RNA alpha material treated with RIII. Thus,we can conclude that alpha process does not make the same dsRNA speciesas equimolar process (FIG. 8B).

dsRNA Abundance by RNase III and Cytokine Data from RP Fractions

A RP Fractionation of hEPO treated with and without RNase III wasperformed. hEPO G5 mRNA transcribed via both equimolar and alphaprocesses were purified by reverse phase HPLC. Fractions were collectedacross the elution gradient. Fractions were treated with RNase IIIsubsequently analyzed by capillary electrophoresis comparing untreatedRNA and RNase III treated (overlaid). The fractions of RNA were alsotransfected into BJ Fibroblasts and IFN-B induction was assessed pre andpost RNase III treatment (FIG. 9).

A Capillary Electrophoresis analysis of RP fractionated hEPO EQ+/−RIIItreatment was performed. hEPO EQ was treated with RNase III then RPpurified, fractionated, and analyzed by CE (blue: RIII treated; black:untreated) Early equimolar fractions (fractions 1-6) containing RNAimpurities denote appreciable abundance of dsRNA/RNase III substrate.dsRNA was enriched in early fractions This was confirmed by high INF-Blevels. Equimolar fractions 7-9 denote lower levels of dsRNA/RNase IIIsubstrate by CE, which was also confirmed by decreasing levels of IFN-B.RNase III treatment of each fraction reduces IFN-B induction to basallevels, which again confirms IFN-B impurities are comprised of dsRNA(FIG. 10A-C). In vitro IFNbeta analysis of hEPO EQ G5 untreated or afterRNase III treatment (FIG. 10D).

A Capillary Electrophoresis analysis of RP fractionated hEPOAlpha+/−RIII treatment was performed. hEPO alpha was treated with RNaseIII then RP purified, fractionated, and analyzed by CE (blue: RIIItreated; black: untreated) (FIG. 11A-C). In vitro IFNbeta analysis ofhEPO Alpha G5 untreated or after RNase III treatment (FIG. 11D). Allfractions denote trace levels of dsRNA (RNase III substrate) by bothcapillary electrophoresis as overlays electropherograms are virtuallyidentical. This was confirmed by basal levels of IFN-B in both treatedand untreated fractions. RNA transcribed with alpha process was devoidof dsRNA, despite non-full length/truncated RNA impurities being presentin early fractions.

ELISA Detection of dsRNA Abundance

The J2/K2 dsRNA ELISA was developed to measure dsRNA abundance. Plateswere coated with J2 monoclonal (IgG) antibodies and then blocked. TheRNA of interest was then added at given concentrations and incubated foran hour. The K2 monoclonal antibody was added (IgM), and anHRP-conjugated anti-IgM goat polyclonal antibody was added. TMB wasadded to develop the signal. The assay detects duplexes greater than 40base pairs in length. J2 can assist in RIII endpoint determination. Aknockdown of dsRNA was observed with RP or RNase III treatment (FIG.12). The J2 assay suggests that there was considerably more dsRNA inequimolar material than alpha. The RP process removes dsRNA materialfrom EQ samples, to a similar extent as RIII treatment. The alphareaction mRNA had less dsRNA in feedstock compared to the equimolar IVTproduct. Also, a knockdown in dT purified RNase III materials wasobserved to be greater than TrisRP. The assay also illustrated thatdsRNA was removed by RNase III treatment (FIG. 13). While dsRNA levelsdetected by J2 vary based on construct/process/chemistry, RNase IIItreatment appears to reduce most dsRNA levels to baseline. LCMS analysisafter Nuclease P1 treatment showed additional NTPs present in FFB mRNA,as reverse complements initiated with non-G were present in higherabundance in the equimolar group, as compared to the alpha reactiongroup (Table 2).

TABLE 2 pppA pppG pppC pppU ng/mL ng/mL ng/mL ng/mL G5 hEPO 31 172 53BQL Equimolar uncapped G5 hEPO 12 159 BQL BQL Formulation of inventionUncapped

Mitigation of RNA Templated Transcription in Low Temperatures IVTs

An IFNbeta response for hEPO construct prepared using differentprocesses and chemistries was performed. As shown in the IVTcharacterization study, alpha reaction was less sensitive to lowtemperature-induced cytokine spikes (FIG. 14). Standard Equimolar IVTwhen performed at 25 C generate enhanced IFN-B inducing impurityabundance. A Total Nucleotide analysis was performed on hEPO constructsprepared using different processes. Alpha process conditions (GDP andGTP) both mitigate extraneous IFN-B inducing impurity formation whenperforming IVT at both 37 C and 25 C. Nucleotide distribution wasconsistent across the control regions in the Nuclease P1 study (greyboxes) and the same hEPO plasmid shows consistent cleavage (FIG. 15).With equimolar IVT processes run at 25 C, abundance of Uridine/1-methylpseudo U was in higher abundance than at 37 C presumably due toevolution of Poly U, Alpha processes (GDP and GTP) shows consistentnucleotide distribution even at 25 C further supporting mitigation ofimpurity formation. The differences in bar height are likely due toconcentration differences. The U content increased over time with 25° C.standard IVTs. The molar corrected nucleotide composition was given inTables 3 and 4. G and U are theoretical values, while A andoverrepresented and C was underrepresented. There was less deviationacross temperature conditions with alpha reaction, and the highestdeviation was observed in the U for standard IVTs.

TABLE 3 % A % G % C % U GDP 37 C. 2 hr 35.5 27.9 18.3 18.3 GDP 25 C. 6hr 35.2 28.1 18.4 18.2 GDP 25 C. O/N 34.8 27.7 19.3 18.2 GTP 37 C. 2 hr35.2 27.5 18.9 18.4 GTP 25 C. 6 hr 35.2 27.5 19.1 18.3 GTP 25 C. O/N35.1 27.7 19.0 18.2 G5 37 C. 2 hr 35.4 26.8 19.5 18.3 G5 25 C. 6 hr 33.827.7 18.3 20.1 G5 25 C. O/N 32.1 26.5 17.1 24.3 G0 37 C. 2 hr 36.5 26.719.1 17.6 G0 25 C. 6 hr 33.0 24.4 17.1 25.6 Theoretical 31.7% 27.3%22.4% 18.6%

TABLE 4 Standard Deviation A G C U GDP 0.34 0.23 0.53 0.04 GTP 0.07 0.150.06 0.09 G5 Std 1.66 0.60 1.20 3.08 G0 Std 2.53 1.65 1.43 5.61

“Forced” RNA Templated Transcription

An impurity analysis was performed by LCMS of RNA-based IVT in differentchemistries using G5 eGFP EQ. IVTs were set up (in differentchemistries) using 4 mg/mL eGFP G5 equi RNA and no DNA template. polyUspecies are generated from RNA-templated transcription. RNA-templatedtranscription occurs independent of chemistry. Reverse phase purifiedeGFP G5 (cold) was incubated at 4 mg/mL (consistent with a typical yieldof an in vitro transcription reaction), with all the IVT componentsexcept DNA template, which was determined to be below threshold requiredfor in vitro transcription by qPCR, to explore the RNA-templatedtranscription phenomenon. The materials were analyzed by LC/MS (FIG. 16A(Equimolar process), 16B (alpha process)).

A IFNbeta analysis for RNA-templated IVT products was performed.Material was from FIG. 16. After RNA-based IVT, samples were analyzed invitro to determine if there was a correlation between LCMS analysis(polyU species) and IFNbeta response. Alpha (A100) RNA-based IVTsproduce material that has a lower CK response than equimolar material.(G0 or G5); Suggestive again that alpha process mitigates the formationof RNA templated transcription products. All G6 was cold; (intrinsicallycold despite formation of impurities) (FIG. 17).

A dsRNA cannot be capped by vaccinia was performed to determine whetherdsRNA can be capped. Forward and reverse complement oligos both with 5′triphosphates were annealed then were subjected to capping process usingvaccinia to determine whether dsRNA could be capped. pppF oligo(contains 5′pppG) can be capped to cap1 using vaccinia. pppRC oligo(contains 5′pppU) cannot be capped using vaccinia. dsRNA with pppF+pppRCcannot be capped using vaccinia (FIG. 18).

A Dephosphorylation of dsRNA using CIP was performed to determine ifcalf intestinal phosphatase dephosphorylate dsRNA with different 5′/3′overhangs? Various F and RC oligos were annealed then treated with CIPand analyzed by LCMS for dephosphorylation efficiency. dsRNA with 5′overhangs can be fully dephosphorylated. perfect duplex dsRNA can bepartially dephosphorylated. dsRNA with 3′ overhangs cannot be fullydephosphorylated. This demonstrates why phosphatase was not 100%effective at reducing CK response (FIG. 19).

A CE Purity of mRNAs dosed in aforementioned in vivo study denoted inelectropherograms was performed. (FIG. 20). Structure may play a role,as the 5′UTR may impact the effectiveness of alpha reaction (FIG. 20).

In Vivo Studies

RIII treatment showed distinct cytokine knockdown in mice, both withunmodified and completely modified (all uridine was1-methylpseudouridine) species. It may be used as a standalone method toknockdown cytokines to near basal levels. Female mice (n=5 per group)were injected once with 0.5 mg/kg of selected test materials.

An IFNbeta analysis of G0 and G5 hEPO mRNA transcribed using differentIVT processes and purification permutations was performed to determinewhat the IFNbeta response for material generated with differentchemistries, processes, and treatments (in vitro; BJ Fibroblasts).+RIII, +RP, and alpha processes have similar effects (reduction) onIFNbeta response in vitro in G5. G0 demonstrates that alpha+RIII wassuperior to equi+RIII (FIG. 21).

An In vivo (Balb-C mice) expression of hEPO prepared using G0 or G5chemistries, equimolar or alpha processes, and RNase III treatment viaELISA was performed to determine how expression affected by hEPOprepared using various processes (chemistry, IVT process, RIIItreatment). Same material as FIG. 12, 20, 21, 22. There are nosignificant differences in hEPO expression using RNA generated bydifferent processes (FIG. 22).

A Cytokine (luminex) panel for in vivo (Balb-C mice) G0 and G5 hEPO mRNAtranscribed using different IVT processes and purification permutationswith and without RNase III treatment was performed to determine with thesame material as FIG. 12, 20, 21, 22. Cytokine analysis after treatmentof Balb-C mice with 0.5 mpk formulated RNA. Untreated alpha material hasa similar CK response as EQ material (G5) in IP10. Alpha process inconjunction with RNase III treatment overall appears to confer less.immunostimulatory activity than the equimolar comparator (+RNase III),especially in G0. RIII treatment reduces CK response (FIG. 23A-23D).

A B cell activation from spleens of Balb-C mice treated with hEPOprepared using G0 or G5 chemistries, equimolar or alpha processes, andRNase III treatment was performed, Activated B cells (CD86+CD69+) wereanalyzed for their response after treatment with hEPO prepared usingvarious processes. B cell activation roughly correlates to cytokine(luminex) panel. RIII treatment reduces the CK response, with respect tothe untreated controls (FIG. 24).

An In vitro analysis of short 5′ triphosphorylated oligos was performedto determine whether short 5′ triphosphorylated oligos stimulate anIFNbeta response in vitro (BJF, IFNbeta). ssRNA and dsRNA <12nucleotides or base-pairs do not stimulate IFNbeta in BJFs (FIG. 25).

An In vitro analysis of polyU and dsRNA standards was performed todetermine whether using 5′ triphosphorylated oligo standards, whatstimulates an IFNbeta response in BJFs. pppF+pppRC dsRNA >20 bp wasimmunostimulatory. 5′ triphosphorylated polyU was not immunostimulatoryas ssRNA or dsRNA <25 nt or bp (FIG. 26).

An In vitro analysis of dsRNA standards with 3′ overhang was performedto determine whether there a 3′ overhang length dependence on IFNbetaresponse for dsRNA standards. The longer the 3′ overhang, the lower theIFNbeta response (FIG. 27).

An In vitro analysis of dsRNA standards with 5′ overhang, perfectduplex, and 3′ overhang of varying lengths was performed to determinewhether an overhang length/identity dependence on IFNbeta response.ssRNA pppRC oligos are hot. dsRNA with 5′ overhang (and 20 bp duplex)are less immunostimulatory than perfect duplex. dsRNA with 3′ overhanghave equivalent or less immunostimulation than perfect duplex. Thelonger the dsRNA duplex, the higher the CK response (FIG. 28).

An In vitro analysis of polyU species was performed to determine whatwas required for polyU species to simulate an IFNbeta response. ssRNApolyU standards with 5′ triphosphate are slightly immunostimulatory. AsdsRNA with OH-F30 (30 bp duplex), there was an additive CK response.However, polyU standards are not immunostimulatory in the presence offull length mRNA (A100) (FIG. 29).

An in vitro analysis of ssRNA oligonucleotide standards was performed todetermine what was the IFNbeta response for ssRNA standards. (F orforward oligos represent abortive or truncated species; R or RC orreverse complement oligos represent reverse complement species generatedby RNA-templated transcription). Forward oligos with 5′ triphosphate aredo not stimulate IFNbeta. Reverse complement oligos with 5′ triphosphatestimulate IFNbeta at >25 nt in length (FIG. 30).

An in vitro analysis of dsRNA oligos standards with different 5′functionalities was performed to determine whether the 5′ functionality(triphosphate vs hydroxyl) affect IFNbeta response. 5′ triphosphate on Foligo and 5′ hydroxyl on RC oligo simulates IFNbeta at >20 bp. 5′triphosphate on F oligo and 5′ triphosphate on RC oligo simulatesIFNbeta at >25 bp. 5′ hydroxyl on F oligo and 5′ triphosphate on RColigo stimulates IFNbeta at >20 bp. Only one trisphosphate was necessaryto simulate IFNbeta at >20 bp (FIG. 31)

An IFNbeta analysis of CIP-treated dsRNA oligo standards was performed.dsRNA oligo standards of varying overhang lengths were treated with CIPand IFNbeta response was analyzed. Samples in which dephosphorylation(i.e. 5′ overhang) was observed by LCMS (FIG. 19) did not stimulateIFNbeta much as the untreated sample. Samples that were notdephosphorylated by CIP (i.e. perfect duplex and 3′ overhang) had nochange in IFNbeta response +/−CIP (FIG. 32).

A ssRNA Impurity Dose Response (IFNbeta in BJ Fibroblasts) was performedto determine what was the dose dependence of ssRNA impurity standards invitro (BJF, IFNbeta). How much of any one type of impurity was requiredto stimulate a response. <20 mer ssRNA (RC) was not hot. >30 mer ssRNA(RC) stimulated IFNbeta at about >1 ng/uL (or >2.5 ng/transfection).There was an apparent length dependence to ssRNA CK response (RC). Thelonger the oligo, the higher the CK response (FIG. 33).

A dsRNA Impurity Dose Response (IFNbeta in BJ Fibroblasts) was performedto determine whether the dose dependence of dsRNA impurity standards invitro (BJF, IFNbeta). 20-30 bp dsRNA stimulates IFNbeta at ˜>1 ng/uL(>2.5 ng/transfection). >30 bp dsRNA stimulates IFNbeta at ˜0.1 ng/uL(>0.25 ng/transfection). >1000× dilution of >30 bp dsRNA was required tosilence IFNbeta response . . . indicating that just a few molecules ofthese impurities was enough to stimulate a response. There was anapparent length dependence to dsRNA CK response . . . the longer theduplex, the higher the CK response (FIG. 34).

An IFNbeta Response for modified 5′ nucleotide on Forward OligoStandards was performed to determine whether the 5′ nucleotide(trisphosphorylated) affect CK response. When a 5′A/C/U was added to theF oligos, was a CK response induced. 5′ non-G does not change IFNbetaresponse (still cold). This suggests that there was a sequencedependence (since RCs are hot) (FIG. 35).

An IFNbeta Response for modified 5′ nucleotide on Reverse ComplementOligo Standards was performed to determine whether the 5′ nucleotide(trisphosphorylated) affect CK response. When a 5′G was added to the RColigos, was a CK response induced. 5′ pppG on RC oligos SILENCES IFNbetaresponse. This suggests that no matter what the sequence identity was, a5′ G will silence the CK response (FIG. 36).

An IFNbeta Response for 5′ hydroxyl functionalized dsRNA was performedto determine how much does the 5′ functionality (ppp vs OH) affectcytokine response in dsRNA. 5′ ppp-F/5′ppp-RC duplex to 5′OH-F/5′OH—RCwere tested. There was an IFNbeta response for >30 bp dsRNA with either5′ ppp or 5′ OH. This suggests that dsRNA >30 bp, no matter what the 5′functionality was, stimulated a IFNbeta response in BJFs (FIG. 37).

Implications

RNA templated transcription as an IVT byproduct was reduced greatly andnearly eliminated with the alpha reaction process. As a result ofmitigating the formation of reverse complements, both impurities ofinterest are addressed: dsRNA (with 5′ppp) and RNAs initiating withnon-pppG (pppA, pppC, and pppU).

RNA templated transcription was enhanced at IVT reaction temperaturesless than 37° C. (for example, at 25° C.). The ramp time to achieve 37°C. heating while IVT reaction was underway, can lead to higher impuritylevels, especially as 25° C. to 37° C. time ramp increases. The alphareaction process was more forgiving at 25° C., as trace RNA templatedtranscription species were detected at ambient temperature.

RP was more effective with alpha reaction than with the equimolarprocess. There was a lower impurity load with alpha reaction, whichleads to improved separations. One can solve for “purity” moreexplicitly and may potentially increase load challenge, increasing theproductivity. A single RP cycle was adequate to knock an unmodifiedspecies to baseline using GDP alpha reaction, while two to threesequence RP cycles are required to get two fractions of an unmodifiedspecies using equimolar IVT.

Example 5. RNA Generated with Alpha Process has <40% Run-on Transcripts

hEPO RNA was generated using equimolar or alpha processes. RNA wasdigested with Rnase T1 and the tail fragment was analyzed by LCMS. Atail fragment with a 3′ mP indicates a run-on transcript. The alphaprocess generated a 3′OH (clean) calculated to be 32780 Da and a5′OH/3′mP (run-on) calculated to be 32860 Da. The equimolar processgenerated a 5′OH/3′mP (run-on) of 32861 Da and a much smaller amount of3′OH (clean) (FIG. 38).

Example 6. Total Digestion Indicates that RNA from Equimolar Process hasa Higher Abundance of Non-GTP 5′ Nucleotide than RNA Made with AlphaProcesses

Short open reading frame RNA was generated using four differentconditions: 37° C. 2 hours (standard) versus 25° C. 6 hours andequimolar versus alpha. Each RNA was enzymatically digested to singlenucleotides, then the 5′ nucleotide abundance was analyzed by LCMS (e.g.pppG or GTP, pppA or ATP, etc.). A 5′ G is the first templatednucleotide, which means that if an impure RNA population was generatedthen there would be a large fraction of 5′ nucleotides as ATP, CTP, orUTP (e.g. equimolar processes), and if a pure RNA population wasgenerated then the majority of 5′ nucleotides would be GTP (e.g. alphaprocesses).

Example 7. Short Open Reading Frame RNA Generated by Alpha ProcessesGenerates Fewer Reverse Complements

Short open reading frame RNA was generated in G0 (wild type) and G5(m¹Ψ) chemistries using equimolar or alpha processes containing either32P-GTP or 32P-CTP. 32P-GTP labels abortive transcripts and 32P-CTPlabels reverse complement transcripts. There was no difference inabortive or RC profiles between G0 and G5 chemistries. Equimolar andalpha processes have similar abortive profiles. The equimolar processgenerates more reverse complements than the alpha process.

Example 8. RNase T1 Digestion Informs Run-on Transcript Populations

RNase T1 is an endonuclease that specifically cleaves RNA afterguanosine nucleotides, leaving a 5′ hydroxide and a 3′ monophosphate.For constructs that contain a templated guanosine nucleotide at the 3′end, RNase T1 can be used to distinguish populations of run-ontranscripts, which leave a 3′ monophosphate, compared to cleantranscripts, which contain a 3′ hydroxide. hEPO RNA was generated usingequimolar or alpha processes then digested with RNase T1 and analyzed bymass spectrometry. The 3′ oligo fragment was analyzed and quantified forits 3′ heterogeneity. RNA generated with equimolar process containsapproximately 70-80% run-on transcripts, while RNA generated with alphaprocess contains 30-40% run-on transcripts.

Example 9. Total Digestion of RNA to Determine Sample Purity

A short model RNA construct (surrogate RNA 1) was generated usingequimolar or alpha processes at 37° C. or 25° C. incubation. Each samplewas purified by oligo dT resin to remove unreacted NTPs. Each sample wasenzymatically digested to single nucleotides using SI and benzonasenucleases. The abundances of 5′ nucleotides, which are triphosphorylatedat the 5′ end, were analyzed by mass spectrometry. Extracted ion peakswere integrated and % NTP abundances are tabulated in Table 5. Since 5′GTP is the first templated nucleotide, a pure RNA population isindicated by a high % GTP value. Likewise, an impure RNA population isindicated by a relatively low abundance of GTP. Samples generated usingthe EQ process have >5% non-GTP 5′ nucleotides, while samples generatedusing alpha process have <5% non-GTP 5′ nucleotides.

TABLE 5 % GTP % ATP % CTP % UTP Surrogate RNA 1 equimolar 94.4 0.8 3.81.2 37° C. Surrogate RNA 1 alpha 37° C. 97.4 0.3 2.3 0.0 Surrogate RNA 1equimolar 68.1 5.0 12.3 21.6 25° C. Surrogate RNA 1 alpha 25° C. 97.60.2 1.9 0.4

Example 10. dsRNA ELISA Indicates Presence of dsRNA

hEPO RNA construct was generated using equimolar or alpha processes andpurified either by oligo dT resin only (−RP) or with reverse phasepurification (+RP). ELISA using dsRNA-specific antibodies is used todetermine relative differences in the purities of RNAs generated usingdifferent processes. RNAs generated using equimolar process containsignificantly more dsRNA than RNAs generated with alpha process (FIG.39). Reverse phase purification improves the purities of −RP RNAs.

Example 11. Radioactive Sequencing Gel Analysis Determines Sample Purity

A short model RNA construct (surrogate RNA 1) was generated usingequimolar or alpha processes. Each IVT reaction contained either³²P-GTP, which labels abortive transcripts, or ³²P-CTP, which labelsreverse complement transcripts. RNA samples were analyzed by sequencinggel. Based on the ³²P-GTP data, there is no difference in abortivetranscript abundance between the two processes. Based on the ³²P-CTPdata, equimolar process generates more reverse complements than alphaprocess.

Example 12. In Vivo Analysis of dsRNA Doped into mRNA

Reverse phase purified hEPO mRNA was doped with 5%, 0.5% or 0.05% w/w 60bp dsRNA, which corresponds to the first 60 nucleotides of the intacthEPO construct. The mRNA samples were formulated in MC3 then dosed by IVinto C57BL/6 and Balb-c mice at 0.5 mpk. After 24 hours, the spleenswere harvested and homogenized to generate single cell suspensions. Thesplenic cells were stained for B cell markers then analyzed by flowcytometry. The activated B cell populations were analyzed based on theirexpression of CD86 and CD69 markers. The 60 bp dsRNA has the followingsequence:

60 mer 5′ UTR Epo F GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGAGUGCACG 60 mer 5′ UTR Epo RCGUGCACUCCCAUGGUGGCUCUUAUAUUUCUUCUUACUCUUCUUUUCUC UCUUAUUUCCC

Groups that received hEPO mRNA generated by alpha process had lower Bcell activation than groups that received hEPO with doped dsRNA. Serumwas also collected at 6 h and analyzed by a cytokine luminex panel. Theexpression trends for IP-10, IFN-gamma, and IFN-alpha markers from theluminex panel were consistent with the trends from the B cell activationanalysis, indicating the dsRNA in the doped samples triggered the type Iinterferon pathway, which led to B cell activation.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

What is claimed is: 1.-115. (canceled)
 116. A method of preparing RNAcomprising (a) forming a reaction mixture comprising a DNA template andNTPs including adenosine triphosphate (ATP), cytidine triphosphate(CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP) or ananalog of each respective nucleotide, and a buffer magnesium-containingbuffer, and (b) incubating the reaction mixture and producing acomposition comprising vitro-transcribed (IVT) RNA, wherein:  1) theconcentration of at least one of GTP, CTP, ATP, and UTP is at least 2×greater than the concentration of any one or more of ATP, CTP or UTP; or 2) the reaction further comprises a nucleotide diphosphate (NDP) or anucleotide analog and wherein the concentration of the NDP or nucleotideanalog is at least 2× greater than the concentration of any one or moreof ATP, CTP or UTP.
 117. The method of claim 116, wherein the ratio ofconcentration of GTP to the concentration of any one ATP, CTP or UTP isat least 2:1 to produce the RNA.
 118. The method of claim 116, whereinthe ratio of concentration of GTP to concentration of ATP, CTP and UTPis 2:1, 4:1 and 4:1, respectively.
 119. The method of claim 116, whereinthe ratio of concentration of GTP to concentration of ATP, CTP and UTPis 3:1, 6:1 and 6:1, respectively.
 120. The method of claim 116, whereinthe reaction mixture comprises GTP and GDP and wherein the ratio ofconcentration of GTP plus GDP to the concentration of any one of ATP,CTP or UTP is at least 2:1.
 121. The method of claim 120, wherein theratio of concentration of GTP plus GDP to concentration of ATP, CTP andUTP is 3:1, 6:1 and 6:1, respectively.
 122. The method of claim 116,wherein the reaction mixture further comprises guanosine diphosphate(GDP).
 123. The method of claim 116, wherein the composition issubstantially free of reverse transcription complement product.
 124. Themethod of claim 116, wherein greater than 90% of the mass of the RNAcomprises single stranded full length transcripts.
 125. The method ofclaim 116, wherein the composition is substantially free of RNAse IIIinsensitive fragments.
 126. The method of claim 116, wherein thecomposition comprises a population of single stranded partial RNAtranscripts in a sense orientation and wherein greater than 80% of thepopulation of single stranded partial RNA transcripts in a senseorientation has a nucleotide length of 100 nucleotides or less.
 127. Themethod of claim 126, wherein greater than 90% of the population ofsingle stranded partial RNA transcripts in a sense orientation has anucleotide length of 100 nucleotides or less.
 128. The method of claim116, wherein less than 0.25% of the mass of the RNA in the compositionis cytokine-inducing RNA contaminant.
 129. The method of claim 128,wherein the composition is substantially free of cytokine-inducing RNAcontaminant.
 130. The method of claim 116, wherein less than 0.5% of themass of the RNA in the composition is reverse complement transcriptionproduct.
 131. The method of claim 130, wherein the reverse complementtranscription product is a dsRNA comprising a strand comprising asequence that is a reverse complement of at least a portion of the IVTRNA or a dsRNA comprising a strand comprising a polyU containingsequence.
 132. The method of claim 131, wherein the strand comprisingthe sequence that is the reverse complement of the IVT RNA or the strandcomprising the polyU sequence initiates with a 5′ triphosphate (5′-PPP).133. The method of claim 130, wherein the reverse complementtranscription product comprises a reverse complement of the 5′-end ofthe IVT RNA and/or a reverse complement of the 3′-end of the RNAencoding the polypeptide of interest.
 134. The method of claim 116,wherein the IVT RNA is an RNA encoding a polypeptide of interest andwherein the reverse complement of the RNA encoding the polypeptide ofinterest comprises a sequence complementary to all or a portion of anopen reading frame of the RNA encoding the polypeptide of interest. 135.An unpurified composition comprising in vitro-transcribed (IVT) RNAproduced by the method of claim 116, wherein the ratio of concentrationof GTP to the concentration of any one ATP, CTP or UTP is at least 5:1,greater than 90% of the mass of the RNA comprises single stranded fulllength transcripts, and the composition is substantially free of reversetranscription complement product and RNAse III insensitive fragments.